Objective To systematically delineate the expression profile of the m⁵C RNA methyltransferase NSUN7 in multiple human and mouse tissues and organs, providing a foundation for investigating its biological functions. Methods A multi-faceted approach was employed, including immunohistochemistry, BioGPS database mining, reverse-transcription quantitative PCR, and Western blot, to analyze the spatial localization and quantitative levels of NSUN7 expression. Results NSUN7 protein was primarily localized in the cytoplasm of human and mouse cells; protein expression was enriched in epithelial cells. NSUN7 demonstrated a complex regulatory pattern in metabolically active organs: a discordance between high mRNA and low protein levels was observed in the liver, while its protein expression was specifically elevated in thermogenic brown adipose tissue compared with white adipose tissue. The strong positive expression of NSUN7 in cervical squamous epithelium aligns with its previously reported pro-oncogenic functions. Critically, NSUN7 in human testis was expressed in spermatogonia and spermatocytes,aligning with genetic studies linking loss-of-function mutations in NSUN7 to impaired spermatogenesis. Conclusions This study provides the first systematic mapping of the NSUN7 expression landscape across multiple human and mouse tissues and organs, providing a physiological context for its known or potential roles in cancer and male reproduction, as well as revealing its underappreciated expression features in key energy metabolism-related tissues. This work lays the comprehensive groundwork for future functional investigations into the multifaceted roles of NSUN7.