The theca interna contains fibroblast-like mesenchymal cells, endothelial cells and immune cells [Endocr Rev. 1985 Summer;6(3):371-99]. It also contains steroidogenic cells that are responsible for androgen production, and as such the theca interna is different to the other ovarian stromal compartments of the ovary. TGFβ-1 is an important growth factor that regulates ovarian stromal compartments [Reprod Sci. 2014 Jan;21(1):20-31] and inhibits ovarian steroidogenesis [Endocrinology. 1990 Dec;127(6):2804-11]. The aim of our study was to determine the effects of TGFβ-1 on the regulation of various extracellular matrix (ECM) genes in bovine thecal cells. Thecal cells from small antral bovine follicles (4-6 mm) were cultured for 4 days and then treated with TGFβ-1 or SB431542 (TGFβ signalling inhibitor) for 2 days. The cells were then harvested for gene expression analyses. TGFβ-1 stimulated the expression of various genes involved in the regulation of collagen fibrillogenesis such as POSTN (P < 0.001), COL1A1 (P < 0.05), TNC (P < 0.05), FN (P < 0.0001), DCN (P < 0.01), FBN1 (P < 0.01) and FBN2 (P < 0.001), but decreased OGN (P < 0.05) and GPC3 (P < 0.05) expression. The expression of LTBP3 and BMP1 was not affected by TGFβ-1. Since the theca interna is a highly vascularised tissue, we also examined the expression of several ECM genes associated with subendothelial basal laminae. TGFβ-1 had no effect on COL4A1, COL4A4 and COL21A1 expression, but significantly reduced LAMA4 (P < 0.0001), FBLN1 (P < 0.01) and FBLN5 (P < 0.05) expression. SB431542 treatment decreased TNC (P < 0.05) and COL4A4 (P < 0.01) expression, suggesting that endogenous TGFβ signalling was active in our cultures. Our observations suggest that TGFβ-1 could affect the matrix associated with the vasculature and the rigidity of the theca interna, and hence is involved in follicular dynamics.