ABSTRACTSpermatogenesis is a highly complex developmental process that typically consists of mitosis, meiosis, and spermiogenesis. DNA/RNA helicase DHX36, a unique guanine-quadruplex (G4) resolvase, plays crucial roles in a variety of biological processes. We previously showed that DHX36 is highly expressed in male germ cells with the highest level in zygotene spermatocytes. Here, we deletedDhx36in advanced germ cells withStra8-GFPCre and found that aDhx36deficiency in the differentiated spermatogonia leads to meiotic defects and abnormal spermiogenesis. These defects in late stages of spermatogenesis arise from dysregulated transcription of G4-harboring genes, which are required for meiosis. Thus, this study reveals thatDhx36plays crucial roles in late stages of spermatogenesis.