Objective: To systematically delineate the expression profile of the m5C RNA methyltransferase NSUN7 in multiple tissues and organs of human and mouse, thereby providing a solid foundation for investigating its biological functions. Methods: A multi-faceted approach was employed, including immunohistochemistry (IHC), BioGPS database mining, RT-qPCR, and Western blotting, to analyze both the spatial localization and quantitative levels of NSUN7 expression. Results: NSUN7 protein was primarily localized in the cytoplasm of human and mouse cells and its protein expression exhibited a marked enrichment in epithelial cells. Notably, 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 to white adipose tissue. Furthermore, 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, a finding that aligns with genetic studies linking loss-of-function mutations in NSUN7 to impaired spermatogenesis. Conclusion: This study provides the first systematic mapping of the NSUN7 expression landscape across multiple tissues and organs of human and mouse, not only providing a physiological context for its known or potential roles in cancer and male reproduction, but also more importantly, 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.