Objective: To investigate the role and molecular mechanism of RNA binding motif single stranded interacting protein 3 (RBMS3) in the proliferation of breast cancer and its molecular mechanism by regulating the stability of cyclin-dependent kinase inhibitor 1A (p21) at the post-transcriptional level. Methods: Colony formation and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assays were used to evaluate cell proliferation ability in vitro. Flow cytometry was applied to analyze cell cycle distribution and apoptosis rate. Xenograft tumor model in nude mice was established to observe the effect of RBMS3 on tumor growth in vivo. The correlation between RBMS3 and p21 expression was detected by Western blot (WB), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC). Furthermore, actinomycin D (Act D) assay was performed to verify the effect of RBMS3 on p21 mRNA stability. RNA immunoprecipitation (RIP) assay, dual-luciferase reporter assay, and rescue experiment were conducted to confirm the direct binding and functional interaction between RBMS3 and p21. Results: Overexpression of RBMS3 inhibited the proliferation of breast cancer cells and the growth of xenograft tumors in nude mice，induced G0/G1 phase cell cycle arrest, and promoted apoptosis. Conversely, knockdown of RBMS3 promoted breast cancer cell proliferation. Furthermore, RBMS3 expression was significantly positively correlated with p21. RBMS3 directly bound to AU-rich elements in the 3' untranslated region of p21 mRNA and enhanced the stability of p21 transcripts. Knockdown of p21 reversed the decrease in breast cancer cell proliferation induced by RBMS3 overexpression. Conclusion: RBMS3 upregulates p21 expression by directly binding to the AREs in the 3′-UTR of p21 and enhancing its mRNA stability, thereby inhibiting the proliferation of breast cancer cells. These findings suggest that RBMS3 may serve as a potential therapeutic target for breast cancer. Keywords: RBMS3, p21, mRNA stability, Breast cancer