Pulmonary fibroblasts have key roles in the formation and maintenance of lung structure and function, and are involved in tissue repair and remodeling. Transforming growth factor‑β1 (TGF‑β1) induces differentiation of fibroblasts into myofibroblasts, the key effector cells in fibrotic states, which are characterized by the expression of α‑smooth muscle actin (α‑SMA) markers. 1α,25‑Dihydroxyvitamin D3 [1,25(OH)2D3] has been implicated in regulating differentiation, and the vitamin D receptor (VDR) may be a regulator of TGF‑β signaling. In addition, there is presently only limited information regarding microRNA (miRNA) regulation of lung fibroblast differentiation. To determine the role of 1,25(OH)2D3 in regulating the differentiation of fibroblasts induced by TGF‑β1 and the functional importance of miR‑27b, cell culture systems, cell transfection and the 3' untranslated region (3'UTR) luciferase assay were employed. 1,25(OH)2D3 inhibited differentiation and downregulated miR‑27b expression in human lung fibroblasts induced by TGF‑β1. In addition, human lung fibroblasts were transfected with miR‑27b mimic or miR‑27b inhibitor, and demonstrated that the overexpression of miR‑27b decreased the VDR protein expression and increased the expression of α‑SMA, while reducing levels of miR‑27b had opposing effects. Finally, the luciferase reporter assays were performed to confirm that miR‑27b directly targeted VDR 3'UTR. Taken together, these results suggest that 1,25(OH)2D3 inhibits lung fibroblast differentiation induced by TGF‑β1 via miR‑27b targeting VDR 3'UTR, which may be used as a novel treatment strategy in differentiation pathways.