Background <p>Mesenchymal stem cells have been shown to attenuate chemotherapy induced premature ovarian failure (POF) in rats, although the underlying molecular mechanisms remain poorly defined. This study was designed to explore the effects of bone marrow derived mesenchymal stem cells (BMSCs) on ovarian stromal cells (SCs) in a POF model, and to determine whether regulator of G protein signaling 3 (RGS3) acts as a key mediator in regulating ovarian stromal fibrosis and follicular angiogenesis, ultimately improving the follicular developmental microenvironment.</p> Methods <p>BMSCs and SCs were isolated from Wistar rats and cultured. A cyclophosphamide (CTX) -induced SCs injury model was established in vitro. BMSCs were co-cultured with injured SCs using transwell inserts. For the in vivo study, 48 female Wistar rats were randomly divided into three groups: control, CTX model, and CTX+BMSCs treatment group. BMSCs were administered via tail vein injection. Ovarian tissue morphology and fibrosis were assessed using hematoxylin and eosin (HE) staining, Masson’s trichrome staining, and transmission electron microscopy. The expression levels of RGS3, VEGF, CD31, TGF-β1, p-Smad2/3, α-SMA, and Vimentin were detected by immunohistochemistry and Western blot. To investigate the regulatory role of RGS3 in the TGF-β1/Smad2/3 pathway and VEGF expression in theca cells (TCs), RGS3 was silenced using siRNA.</p> Results <p>In vitro experiments showed that CTX treatment significantly downregulated the expression of RGS3 and VEGF in SCs, while upregulating TGF-β1 expression. These changes were reversed by co-culture with BMSCs. In vivo experiments revealed that CTX-induced POF rats exhibited irregular estrous cycles, a reduced number of follicles, and increased deposition of interstitial collagen fibers. Consistently, the expression levels of α-SMA and Vimentin were increased, while RGS3, VEGF, and CD31 were decreased. The TGF-β1/Smad2/3 pathway was also aberrantly activated. BMSCs transplantation significantly improved ovarian morphology, reduced collagen deposition, and restored the regularity of the estrous cycle. Furthermore, BMSCs transplantation reversed the CTX-induced protein expression changes by upregulating RGS3, VEGF, and CD31, and inhibiting the activation of the TGF-β1/Smad2/3 pathway. Finally, silencing RGS3 with siRNA exacerbated the CTX-induced effects in theca cells (TCs), leading to a further decrease in VEGF expression and a further increase in TGF-β1/Smad2/3 expression.</p> Conclusions <p>BMSCs may target RGS3 to coordinately regulate the expression of TGF-β1/Smad2/3 and VEGF. This regulatory axis modulates SCs differentiation and angiogenesis, reduces ovarian stromal fibrosis, and improves the ovarian developmental microenvironment.</p>