Objective: The present study aimed to investigate the effect of seasonal variation on neurotransmitter release in the hippocampus of normal rats and rats with pineal excision. Methods: Two time points, the summer and winter solstice, which are the longest and shortest days of the year, respectively, were selected. Male Sprague–Dawley rats that underwent a sham operation without pineal excision were included as a control group. The concentrations of 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) were determined by radioimmunoassays and enzyme-linked immunosorbent assays, respectively. Results: In the winter, the 5-HT and GABA levels in normal rats exhibited a significant difference compared with those in the operation group (P < .01). A difference was also noted in GABA levels between the normal group and the sham operation group (P < .05). The concentrations of 5-HT and GABA in the hippocampal tissues of the normal group exhibited a seasonal rhythm consisting of elevation during the summer and reduction during the winter (P < .01), while the GABA levels in the sham operation group exhibited a significant difference, with elevation during the summer and reduction during the winter (P < .01). In the operation group, GABA showed the same trend (P < .01). Conclusion: The seasonal rhythm of neurotransmitter secretion by the hippocampus (5-HT and GABA) consisted of elevation during the summer and reduction during the winter. During the winter, the pineal gland exhibited a reverse regulatory effect on the secretion of 5-HT and GABA in the hippocampus, and it exhibited seasonal selectivity with regard to the regulation of 5-HT.