To investigate the transdifferentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into salivary gland-like cells via a novel culture method employing induction culture medium collected from salivary gland cells. Primary salivary gland cells were cultured, and after the first passage, the culture medium was collected for use as induction medium. BMSCs (passage 3) were cultured in either induction medium (induction group) or DMEM/F12 medium with 10 % (v/v) fetal bovine serum (control group) before seeding on three-dimensional collagen/chitosan scaffolds and subcutaneous transplantation into nude mice. The in vitro and in vivo transdifferentiation of BMSCs into salivary gland-like cells was evaluated by immunocytochemical analysis of alpha-amylase and cytokeratin-8 (CK-8) expression. Salivary gland-like cells cultured using this novel method maintained excellent biostability and exhibited relatively stable expression of alpha-amylase and CK-8 in vitro and in vivo. This novel culture method is feasible for inducing the transdifferentiation of BMSCs into salivary gland-like cells.