Melatonin is important for seasonal breeding animals to display their breeding characteristics in an appropriate season by regulating the hypothalamic-pituitary-gonadal axis. The Giant Panda (Ailuropoda melanoleuca) is a seasonal breeding mammal, usually breeding from March to May. [Objectives] To explore the seasonal changes in melatonin and testosterone in male giant pandas, [Methods] urine samples from three adult male giant pandas housed at the Chengdu Research Base of Giant Panda Breeding were collected once a week for one year (2018.8-2019.7) under natural photoperiod conditions. Depending on the local climate, spring is from March to May, summer includes June and July, autumn is from August to November, and winter is from December to February of the following year. These samples were analyzed for urinary melatonin and testosterone levels by ELISA. The seasonal changes of melatonin and testosterone levels were analyzed by one one-way ANOVA, and the differences of inter-seasonal data were analyzed by independent sample t-test. The data of testosterone levels were adjusted for homogeneity of variance by using the log value for analysis, however, all graphs were expressed in the actual testosterone levels. [Results] The results indicated that melatonin levels showed a seasonal secretion pattern (Fig. 1 and 2) and its level in winter was extremely significantly higher than that in in autumn, spring and summer, respectively (P < 0.01), and there were no significant differences among the levels in autumn, spring or summer (P > 0.05). Testosterone levels also showed a seasonal secretion pattern (Fig. 3 and 4) and its level in winter was extremely significantly higher than that in autumn or summer (P < 0.01). Testosterone level in spring was also extremely significantly higher than that in autumn or summer (P < 0.01), however no significant difference was detected between winter and spring (P > 0.05). Additionally, there was no significant difference between autumn and summer either (P > 0.05). [Conclusion] Urinary melatonin and testosterone levels show seasonal changes. Melatonin and testosterone levels increase synchronously from autumn to winter; melatonin level decreases sharply, while testosterone remains at a high level from winter to spring. The results provide reference data for further investigation of the effect of melatonin on the seasonal breeding of giant pandas.