Muscarinic acetylcholine receptor (mAChR) antagonists have been reported to decrease male fertility; however, the roles of mAChRs in spermatogenesis and the underlying mechanisms are not understood yet. During spermatogenesis, extensive remodeling between Sertoli cells and/or germ cells interfaces takes place to accommodate the transport of developing germ cells across the blood-testis barrier (BTB) and adluminal compartment. The cell–cell junctions play a vital role in the spermatogenesis process. This study used ICR male mice and spermatogonial cells (C18-4) and Sertoli cells (TM-4). shRNA of control or M5 gene was injected into 5-week-old ICR mice testes. Ten days post-viral grafting, mice were deeply anesthetized with pentobarbital and the testes were collected. One testicle was fresh frozen for RNA-seq analysis or Western blotting (WB). The second testicle was fixed for immunofluorescence staining (IHF). C18-4 or TM-4 cells were treated with shRNA of control or M5 gene. Then, the cells were collected for RNA-seq analysis, WB, or IHF. Knockdown of mAChR M5 disrupted mouse spermatogenesis and damaged the actin-based cytoskeleton and many types of junction proteins in both Sertoli cells and germ cells. M5 knockdown decreased Phldb2 expression in both germ cells and Sertoli cells which suggested that Phldb2 may be involved in cytoskeleton and cell–cell junction formation to regulate spermatogenesis. Our investigation has elucidated a novel role for mAChR M5 in the regulation of spermatogenesis through the interactions of Phldb2 and cell–cell junctions. M5 may be an attractive future therapeutic target in the treatment of male reproductive disorders.