Interactions between tumors and mesenchymal stem cells (MSCs) can regulate cancer cell behavior and cancer progression. Rat bone marrow-derived MSCs (rMSCs) were isolated and purified by Percoll density gradient centrifugation. Conditioned media from rMSCs (MSC-CM) was prepared, and its role in cancer cell migration and the underlying molecular mechanism were investigated. MSC-CM increased the migration and up-regulated the expression of CXC chemokine receptor 4 (CXCR4) in rat hepatoma cells (CBRH-7919). F-actin remodeling was observed, and the Young's modulus was decreased in CBRH-7919 cells. A CXCR4 inhibitor suppressed the MSC-CM-induced CXCR4 expression and migration, restored the decrease in the Young's modulus and disrupted the formation of F-actin. MSC-CM thus promotes CBRH-7919 cell migration by lessening cell stiffness and increasing F-actin formation through up-regulation of CXCR4 expression. MSC-CM may therefore have a positive impact on cancer metastases and underlines a potential safety issue associated with clinical applications of MSCs.