Abstract: To investigate the effects of photoperiod on the feeding rhythms and gastrointestinal evacuation dynamics of second-instar juvenile Tachypleus tridentatus, this study examined diel feeding rhythms in second-instar juvenile T. tridentatus using eight observation time points over 24h under three photoperiod regimes: natural photoperiod, constant light, and constant darkness. Feeding rhythms were assessed via gastrointestinal satiety indices, and evacuation dynamics were tracked for 24h after satiation. The results showed that there were no significant differences in average satiety among time points within any photoperiod at 3h or 6h post-feeding. Under natural photoperiod at 3h, satiety was significantly higher at night than during the day, whereas no clear diel differences were observed under continuous light or darkness. By contrast, at 6h post-feeding, satiety indices were consistently higher at night than during the day under all three photoperiods. Feeding peaks under the natural photoperiod at 3h post-feeding occurred during the night-time period (21:00—06:00), while no distinct diel feeding rhythm was detected under continuous light or darkness. However, at 6h post-feeding, clear feeding rhythms emerged under both continuous light and continuous darkness, with feeding peaks occurring at 21:00—09:00, and 21:00—06:00, respectively. Most juveniles achieved substantial food intake within 3h, with only marginal increases observed by 6h. Gastrointestinal evacuation exhibited a biphasic pattern characterized by an initial rapid phase followed by a slower phase. First-time feeding juveniles reached 50% evacuation at 14.7h and 80% at 27.1h after satiation, whereas non-first-time feeding juveniles reached the same benchmarks at 8.8h and 16.3h, respectively. These findings demonstrate that second-instar juvenile T. tridentatus exhibit a pronounced diel feeding rhythm, only weakly influenced by photoperiod. Based on these results, it is recommended that juvenile rearing be conducted under a natural photoperiod with at least one feeding event after dusk and moderately extended during the early feeding stage. This study provides a scientific basis for optimizing feeding strategies in the artificial culture of juvenile T. tridentatus.