Human adipose tissue-derived mesenchymal stem cells (AT-MSC) are considered to be a promising source of autologous stem cells in personalized cell-based therapies. Tumor tracking properties of MSC provide an attractive opportunity for targeted transgene delivery into the sites of tumor formation. In the present study, we addressed whether the suicide gene introduction into human AT-MSC could produce a tumor-specific prodrug converting cellular vehicle for targeted chemotherapy. We prepared yeast fusion cytosine deaminase::uracil phosphoribosyltransferase gene-expressing cells [cytosine deaminase (CD)-expressing AT-MSC (CD-AT-MSC)] by retrovirus transduction. We explored their therapeutic potential on a model of human colon cancer in the presence of prodrug 5-fluorocytosine (5-FC). Gene manipulation of human AT-MSC did not sensitize CD-AT-MSC to 5-FC, thus overcoming the inherent disadvantage of suicide effect on cellular vehicle. CD-AT-MSC in combination with 5-FC augmented the bystander effect and selective cytotoxicity on target tumor cells HT-29 in direct coculture in vitro. We confirmed directed migration ability of AT-MSC and CD-AT-MSC toward tumor cells HT-29 in vitro. Moreover, we achieved significant inhibition of s.c. tumor xenograft growth by s.c. or i.v. administered CD-AT-MSC in immunocompromised mice treated with 5-FC. We confirmed the ability of CD-AT-MSC to deliver the CD transgene to the site of tumor formation and mediate strong antitumor effect in vivo. Taken together, these data characterize MSC derived from adipose tissue as suitable delivery vehicles for prodrug converting gene and show their utility for a personalized cell-based targeted cancer gene therapy.