Wolffian duct maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in Wolffian duct development is still undefined. By designing a mesenchyme-specific Ar knockout (AR^cKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal Wolffian ducts and cystic formation at the cranial Wolffian ducts. Ex vivo culture of AR^cKO Wolffian ducts invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote Wolffian duct survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA-seq of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2 and Lamc2) were associated with morphological and cellular changes in AR^cKO male embryos (i.e. reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in AR^cKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in Wolffian duct maintenance and morphogenesis in mice.