Background <p>Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by joint damage and disrupted vitamin D signaling, leading to calcium deposition in joints. This study explores the therapeutic potential of Bavachin (BVN), a phytoestrogen from Psoralea corylifolia, in modulating vitamin D signaling in RA.</p> Methods <p>Vitamin D receptor (VDR) structure was modeled using SWISS-MODEL, AlphaFold, and I-TASSER, followed by docking with BVN and Estradiol (E2) via AutoDock Vina. BVN’s effects on VDR mRNA and protein levels in RA-FLS were assessed by qRT-PCR and Western blot(WB). VDR-related BVN targets in RA were explored through protein-protein interaction (PPI) network and pathway analysis in Cytoscape. BVN’s impact on RXRα expression and VDR–RXRα interaction was examined by WB and immunofluorescence. Alizarin staining evaluated calcium deposition, while qRT-PCR analyzed BVN’s regulation of calcium-binding proteins.</p> Results <p>In silico analysis revealed a strong interaction of VDR with BVN with Gibbs-free energy of -7.2 Kcal/mol with prominent H-bonds. Further, in vitro study in RA-FLS revealed that BVN treatment increased VDR mRNA and protein expression. PPI and pathway enrichment analysis retrieved RXRα as the prominent protein to be targeted by BVN in Vitamin D signaling. BVN treatment also significantly upregulated RXRα expression and enhanced the interaction between VDR and RXRα. Further, BVN modulated associated calcium signaling, reduced calcium deposition in RA-FLS and significantly downregulated calcium-binding proteins CALB1, CALB2, NCX1, TRPV5, and TRPV6.</p> Conclusion <p>Collectively, this study depicted a prominent therapeutic efficacy of BVN in targeting Vitamin D signaling and associated calcium deposition to alleviate RA pathogenesis.</p>