Abstract: This study aims to investigate the variation patterns of bitterness in the fresh-squeezed Shitougan juice under different storage conditions. A 4-factor 2-level full factorial experiment was also implemented using single-factor optimizations. The content of primary bitter compounds and Bitterness were used to take the after-bitterness values as evaluation criteria. The performance of the pasteurization was examined in the light exposure, temperature, and air percentage. The analysis of variance (ANOVA) and correlation analysis were carried out to calculate the Dot values. A systematic investigation was made to clarify the influence of different storage conditions on the content of major bitter compounds and the perception of bitterness in the juice. There was a relationship between these compounds and the bitterness perception of Shitougan juice. The results showed that the Limonin (19.66 μg/mL), Naringin (320.61 μg/mL), Neohesperidin (371.35 μg/mL), and Arginine (50.78 mg/100 mL) exhibited significantly (P<0.05) higher concentrations in Shitougan juice subjected to pasteurization at 70°C for 10 min, compared with the non-pasteurized juice. Storage in darkness significantly increased the contents of Limonin, Naringin, Neohesperidin, and Arginine (in non-pasteurized juice) (P<0.05). Exposure to light within a specific intensity elevated the concentrations of Limonin, Naringin, and Neohesperidin in juice. In unpasteurized juice, the light intensity significantly (P<0.05) suppressed the three compounds. Conversely, the higher light intensities markedly increased their concentrations in pasteurized juice under illuminated conditions. During storage of pasteurized Shitougan juice, the concentrations of three bitter compounds—Limonin, Naringin, and Neohesperidin—showed a significant increasing trend with the elevated ambient temperatures. Elevated air percentage significantly (P<0.05) increased the concentrations of Limonin, Naringin, and Neohesperidin in the fresh Shitougan juice. Also, there was the increase in the limonin content of the pasteurized juice; Once the air percentage reached 90%, their peak concentrations were 20.52 μg/mL (Limonin in fresh juice), 322.35 μg/mL (Naringin), 374.38 μg/mL (Neohesperidin), and 20.75 μg/mL (Limonin in pasteurized juice), respectively; However, there was the significant (P<0.05) decrease in the Naringin and Neohesperidin contents in juice after pasteurization. Arginine content also demonstrated the exceptional stability, indicating negligible fluctuations, in response to the variations in light exposure, temperature fluctuations, or alterations in the air ratio under the experimental conditions. The four factors—light intensity, temperature, air percentage, and pasteurization status—as well as their interaction, exhibited the substantial variations in the degree of impact on both the concentrations of different bitter compounds in the juice and the sensory attributes of bitterness intensity and After-Bitterness scores. Air percentage exerted the strongest influence on Limonin content; The interaction between air percentage and pasteurization status was most significantly impacted on Naringin levels; Whereas the pasteurization status shared the dominant effect on Neohesperidin and Arginine concentrations. The interaction between light and pasteurization status constituted the primary determinant for juice Bitterness, whereas the temperature solely governed the intensity of After-Bitterness. In all experimentally treated juice samples, the four bitter compounds collectively shared statistically significant contributions to the bitterness intensity. Their contribution hierarchy was established as follows: Limonin > Arginine > Naringin > Neohesperidin. After-Bitterness with a characteristic sensory was presented in the freshly processed and stored Shitougan juice, with the juice bitterness intensity proportionally to the augmentation of After-Bitterness. Limonin served as the primary mediator of After-Bitterness expression, whereas the increasing concentrations of Naringin and Neohesperidin induced the moderate attenuation of this lingering sensation. Vacuum packaging was implemented to combine with the low-temperature environments during processing and storage. The accumulation of Limonin in juice was effectively inhibited to mitigate the intensification of both primary Bitterness and After-Bitterness. The finding can be expected to elucidate the effects of light exposure, ambient temperature, headspace ratio, and pasteurization on the bitter compounds and bitterness perception during citrus juice production. As such, the scientifically grounded theoretical framework was established to optimize the sensory quality control in citrus juice processing.