Testicular heat stress is a critical factor contributing to male infertility, with spermatocytes exhibiting heightened sensitivity to temperature elevation. This study systematically elucidates the protective mechanisms of melatonin against heat stress-induced spermatocyte injury. In a murine heat stress model, melatonin intervention significantly reduced testicular accumulation of malondialdehyde (MDA) induced by heat stress, enhanced the activities of catalase (CAT) and superoxide dismutase (SOD), and suppressed germ cell apoptosis by downregulating the pro-apoptotic protein Bax and upregulating GPX4 expression. Sycp3 immunohistochemistry demonstrated that melatonin significantly improved spermatocyte structural integrity. In the GC-2spd (ts) spermatocyte cell line model, melatonin treatment markedly reduced MDA levels and alleviated heat stress-induced oxidative apoptosis and proliferation inhibition by downregulating key apoptotic proteins (Bax, Caspase3, and cleaved-Caspase3). Mechanistic studies revealed that melatonin restores autophagic balance by modulating the expression of autophagy-related proteins LC3-I, LC3-II, and P62. Concurrently, melatonin downregulated ferroptosis markers P53 and COX2, inhibiting ferroptosis by blocking DNA damage response and inflammatory amplification pathways. Melatonin synergistically maintained cellular redox homeostasis by downregulating the NRF2/HO-1 pathway and upregulating GPX4 expression, significantly reducing Fe²⁺ accumulation and ameliorating iron metabolism dysregulation. This study unveils the molecular mechanisms by which melatonin mitigates testicular heat stress injury through a multi-target regulatory network, providing novel therapeutic strategies for clinical intervention in heat stress-associated infertility.