RAD54B, a human paralog of RAD54 L, belongs to the SW12/SNF2 superfamily of helicases. Up to now, few studies have reported the role of RAD54B in breast cancer (BC). In this study, we reported that RAD54B was amplified and the expression of RAD54B was elevated in BC, as identified through a bioinformatics analysis of open databases (cBioPortal and Oncomine) and an immunohistochemical analysis of a tissue microarray (TMA). Analysis of Kaplan-Meier plotter (KM plotter) showed that high RAD54B transcription activity was critically linked with poor prognosis of BC patients, in particular those with the luminal A subtype. Gene set enrichment analysis (GSEA) performed on dataset GSE1456 demonstrated that gene expression signatures associated with survival, proliferation, cell cycle, apoptosis and p53 signaling were crucially enriched, when the level of RAD54B was elevated. Further, gain-of-function studies were conducted on luminal A BC cell lines MCF-7 and ZR-751 to validate these findings. Consistently, RAD54B knockdown suppressed cell proliferation in vitro, as well as delayed tumor growth in vivo. Collectively, our results address the biological role of RAD54B in the neoplastic process of luminal A BC. Bioinformatics analysis, additionally, indicates RAD54B with a predictive value for this BC subtype.