Abstract: Photoperiod serves as an essential environmental cue that facilitates seasonal acclimatization and thermoregulation in birds. However, its effects on basal and substrate metabolism in Zebra Finches (Taeniopygia guttata) remain unclear. To explore the influence of photoperiod on basal metabolism and substrate metabolism in Zebra Finches, basal metabolic rate (BMR), body mass, cellular metabolic activities, and substrate metabolism were investigated under different photoperiods. After one week of exposure to a short photoperiod, Zebra Finches exhibited a temporary decrease in BMR, gross energy intake, digestible energy intake, and digestibility, although body mass remained unchanged throughout the experiment. After four weeks of acclimation, no significant differences were observed among different groups in state 4 respiration, cytochrome c oxidase activity, citrate synthase activity, avian uncoupling protein expression, or circulating triiodothyronine and thyroxine hormone levels. In terms of substrate metabolism, short photoperiod-exposed finches showed increased pectoral muscle glycogen content and elevated serum triglyceride and free fatty acid levels, accompanied by a decrease in body fat. No differences were detected in serum glucose levels or in the activity and mRNA levels of carnitine palmityl-transferase-1 and β-hydroxyacyl Co-A dehydrogenase. These findings suggest that changes in photoperiod may serve as signals for substrate metabolism remodeling, while having only transient effects on basal metabolism in Zebra Finches.