Renal cell carcinoma (RCC) ranks among the most prevalent malignancies of the genitourinary system, with a steadily rising incidence. Despite growing attention, the etiology and underlying mechanisms of RCC remain incompletely understood. Epigenetic modifications, particularly DNA methylation, have emerged as critical regulators in various malignancies, including RCC. Sperm-associated antigen 6 (SPAG6), initially identified in human testicular tissue and considered a marker for testicular tumors, has been associated with the pathophysiology of several malignancies. This study aimed to elucidate the role of aberrant SPAG6 methylation in RCC progression.We first analyzed SPAG6 expression and methylation patterns in RCC and adjacent normal tissues using data from The Cancer Genome Atlas (TCGA) and the Epigenome-Wide Association Study (EWAS) databases. Clinical tissue specimens from Peking University First Hospital were then examined to explore the association between SPAG6 expression/methylation and the clinicopathological features of RCC patients. The correlation between SPAG6 expression and promoter methylation was further validated in RCC cell lines. Functional roles of SPAG6 in cell proliferation, invasion, cell cycle regulation, and apoptosis were investigated through in vitro cellular assays and in vivo xenograft models. Finally, transcriptome sequencing was performed to explore the molecular mechanisms by which SPAG6 affects RCC development.SPAG6 expression was markedly downregulated in RCC tissues compared to adjacent non-tumorous counterparts, because of promoter CpG hypermethylation. SPAG6 expression was associated with tumor stage in RCC patients. Functional assays demonstrated that SPAG6 suppresses RCC cell proliferation, invasion, and cell cycle progression, while promoting apoptosis. Mechanistically, SPAG6 inhibited RCC progression by negatively regulating the PI3K/AKT/mTOR signaling pathway.SPAG6 functions as a tumor suppressor in RCC, with its silencing driven by promoter hypermethylation. Through modulation of the PI3K/AKT/mTOR pathway, SPAG6 plays a vital role in restraining RCC initiation and progression.Copyright: © 2025 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.