Spermatogenesis is a complex process requiring precisely controlled metabolic adaptations. Although the genetic and cellular aspects of spermatogenesis have been extensively studied, the underlying metabolic mechanisms remain largely underexplored. In this study, we utilized STA-PUT technology to separate three key cell types involved in mouse spermatogenesis: pachytene spermatocytes (PAC), round spermatids (RS), and elongated spermatids (ES). A comprehensive untargeted metabolomic analysis revealed significant metabolic changes during spermatogenesis, such as reduced methylation-related metabolites and increased glycolytic intermediates and TCA cycle metabolites during ES. Moreover, metabolic differences between germ cells and somatic cells (Leydig and Sertoli cells) were highlighted, particularly in steroidogenesis and lipid metabolism. To investigate clinical relevance, we analyzed human seminal plasma. Samples from individuals with azoospermia displayed significant metabolic abnormalities, including reduced methionine, tryptophan, and arginine, which play vital roles in sperm development. Pathway enrichment analysis revealed disturbances in the metabolism of nucleotide, amino acid, and energy in azoospermia, suggesting potential biomarkers of male infertility. Our findings provide a comprehensive metabolic profile of spermatogenesis and suggest that metabolic alterations may be significant contributors to male infertility, particularly in cases of azoospermia.