Rising global temperatures threaten male fertility, yet the molecular mechanisms by which heat stress disrupts spermatogenesis remain incompletely understood. We employed an integrative multi-omics approach incorporating transcriptomics, proteomics, and metabolomics to examine the impact of sub-lethal HS (Heat stress) on the testes and spermatozoa of male mice. Our results demonstrated that HS significantly diminished sperm motility and compromised the architecture of seminiferous tubules. Multi-omics integration demonstrated a synchronized downregulation of pathways regulating glycolysis, flagellar assembly, and antioxidant defense, as well as disruptions in steroid hormone production and redox equilibrium. These systemic changes collectively contribute to defective energy metabolism, aberrant sperm viability, and diminished tolerance to oxidative stress, ultimately leading to reduced reproductive potential. This study provides an integrated multi-omics view of heat stress–associated molecular disruptions in mouse testes and highlights convergent pathways linked to impaired male reproductive function