NSUN7 is a catalytically inactive RNA m5C methyltransferase essential for sperm flagellum assembly.

RNA 5-methylcytosine (m5C) is a widespread RNA modification mainly catalyzed by the NSUN family, whose dysfunction is linked to various diseases. Among NSUN1-7, all possessing motifs IV and VI essential for RNA m5C formation, NSUN7's catalytic activity and physiological role remain unclear, despite its known association with male infertility. Here, we show that NSUN7 is predominantly enriched in adult mouse testes, particularly in elongated spermatids. Through transcriptome-wide m5C mass spectrometry and ultrafast bisulfite sequencing, we found that Nsun7 knockout has no impact on RNA m5C modification. Mechanistically, compared to NSUN1-6, NSUN7 lacks S-adenosylmethionine (SAM) binding ability due to sequence variations in motif IV, where the key SAM-binding Asp in motif IV is replaced by Leu, thereby abolishing its catalytic activity. Nsun7 deficiency impairs sperm progressive motility, accompanied by defects in axonemes and mispositioning of longitudinal columns. Single-cell RNA sequencing shows that Nsun7 knockout decreases the levels of a cohort of mRNAs related to cilium organization in elongated spermatids. Collectively, our findings resolve the long-standing question of NSUN7's enzymatic role and reveal a non-catalytic regulatory function, providing a more complete understanding of NSUN family biology.