N-glycosylation protein modifications play a crucial regulatory role in numerous biological processes, although their contribution to male reproduction in mammals remains largely undefined. Here, we found that Ribophorin I (RPN1), a subunit of oligosaccharyltransferase complex, is indispensable for spermatogenesis in male germ cells. Germ cell-specific Rpn1 knockout results in significant inhibition of the initiation and progression of meiosis, consequently disrupting homologous chromosome pairing, meiotic recombination, and DNA double-strand break repair during meiosis. N-glycoproteomic profiling revealed that glycosylation levels are reduced in mitochondria-associated proteins, while functional analyses showed that Rpn1 deficiency could inhibit mitochondrial function and abundance, decreasing spermatocyte populations, and potentially reducing energy availability during meiosis and increasing apoptosis levels in mice. These findings highlight the essential physiological functions of N-glycosylation modification in male spermatogenesis and expand our understanding of its role in male fertility.