[Objectives] Melatonin (MEL) is a molecule that conveys photoperiodic information in animals, which is also involved in the regulation of energy homeostasis. The present study aims to investigate the effects of exogenous melatonin on body mass, energy metabolism and thermoregulation in Eothenomys miletus. [Methods] E. miletus were placed at 25 ± 1 ℃ with a photoperiod of 12 L∶12 D (day and night 12 hours each) and received intraperitoneal injection of melatonin (20 μg/kg) daily for 28 days. Body mass, body temperature, food intake, water intake, thermogenic capacity and hormone concentrations were measured. Continuous changes in body temperature, food intake, water intake, resting metabolic rate (RMR) and nonshivering thermogenesis (NST) were measured by repeated measures covariance analysis (with body mass as a covariate). Body mass changes during acclimation were analyzed by repeated measurements, and body mass differences between groups were analyzed by independent sample t test. Differences of serum hormone contents, protein content in liver and brown adipose tissue (BAT), enzyme activity and other indicators between groups were analyzed by covariance analysis (with body mass as a covariate). The relationship between uncoupling protein 1 (UCP1) content, thyroxin 5′-deiodinase (T45′-DII) activity and serum triiodothyronine (T3) content was analyzed by Pearson correlation analysis. [Results] The results showed that exogenous melatonin injection significantly reduced body mass and food intake (Fig. 1a, Fig. 3a), while water intake increased (Fig. 3b). Core body temperature and interscapular skin temperature increased significantly (Fig. 1b, Fig. 2), resting metabolic rate and nonshivering thermogenesis also increased significantly (Fig. 4a, Fig. 4b). Mitochondrial protein content and cytochrome c oxidase (COX) activity in liver and brown adipose tissue increased, there were also significant increases in total protein in brown adipose tissue, uncoupling protein 1, α-glycerophosphate oxidase (α-PGO) and thyroxin 5’-deiodinase activities, but there were no significant effects on total liver protein or α-glycerophosphate oxidase in liver. Serum concentrations of leptin and triiodothyronine were significantly increased, while thyroxine (T4) was significantly decreased. Moreover, testicular mass was significantly reduced after melatonin injection (Table 2), suggesting that melatonin caused gonadal degeneration in E. miletus. Correlation analysis showed a positive correlation between uncoupling protein 1 content and triiodothyronine concentration (Fig. 5a), and a positive correlation between thyroxin 5′-deiodinase activity and triiodothyronine content (Fig. 5b), suggesting that thyroxin 5’-deiodinase may play an important role in the melatonin-induced thermogenesis of brown adipose tissue. [Conclusion] In conclusion, exogenous melatonin may reduce body mass in E. miletus by inhibiting feeding and increasing thermogenesis.