The Amur sleeper (Percottus glenii), a freeze tolerant fish species, can survive being frozen in ice, survival after recovery from freezing. This study investigated the role of melatonin in the recovery of Perccottus glenni following freezing. The fish were categorized into the following groups: non-treated control (Con), pinealectomy (Px), melatonin injection (Mlt), sham control (Sham), and saline injection control (Sal). The results revealed that the melatonin levels were affected by pinealectomy and melatonin injection. The liver and brain of the Px and Mlt groups exhibited autophagy after 4 h, along with the differential expression of endoplasmic reticulum stress-related genes. Furthermore, the activities of the mitochondrial complex initially increased at 4 h and then decreased by 12 h in the Px and Mlt groups, while antioxidant enzyme activities varied across groups and time points. These findings indicated that melatonin plays a key role in the recovery of P. glenni in a time-dependent manner, affecting autophagy, mitochondrial function, and antioxidant capacity. This study is the first to demonstrate melatonin's time-dependent role in facilitating the recovery of P. glenni after freezing, highlighting its critical involvement in modulating autophagy, mitochondrial function, and antioxidant processes. These findings shed light on the physiological mechanisms underlying freeze tolerance and recovery in fish, offering valuable insights for understanding and potentially enhancing the recovery processes in other species.