Abstract: To investigate the photoisomerization mechanism of astaxanthin (AST), the types and contents of Z-configuration in photocatalytic conversion of AST were analyzed by HPLC and Q value method. Additionally, the AST configuration was optimized and frequency analysis was performed using density functional theory (DFT). Thermodynamic, kinetic, and energetic parameters of all-E- and Z-AST isomers were further calculated. The results demonstrated that 9-Z- and 13-Z-isomers were predominantly generated under photocatalysis, with the 13-Z content (17.72%) being significantly higher than 9-Z (13.57%) (P<0.05). Upon illumination, neutral AST molecules generated diradicals, facilitating bond twisting. All-E AST exhibited the smallest frontier orbital energy gap, promoting its transition from the ground state to the excited state, and subsequent isomerization into other configurations. Gibbs free energy analysis revealed the following stability order of AST isomers: all-E>9-Z>13-Z>15-Z, basically consistent with experimental observations. Moreover, the activation energy for all-E→13-Z conversion was higher than for all-E→15-Z/9-Z, while single-Z→double-Z isomerization required significantly higher energy than all-E→mono-Z conversion. Notably, the 13-Z-isomer formation rate was 10-fold and 100-fold higher than that of 15-Z and 9-Z, respectively. The negligible tunneling effect during mutual transformation explained the scarcity of multi-Z-isomers in experiments and the predominance of 13-Z over 9-Z AST. Collectively, these findings elucidate the controlled generation mechanism of AST isomers and establish a theoretical framework for carotenoid isomerization.