We constructed a melanoma-specific oncolytic adenoviral vector Ad.MCDIRESE1.71Hsp3, in which the cytosine deaminase and adenoviral E1A genes linked by the IRES sequence were under the control of a mouse tyrosinase enhancer/promoter transcriptional element in the E1 region of the vector. We also inserted the human heat shock protein 70 (Hsp70) gene driven by the cytomegalovirus promoter into the E3 region of this vector. The RGD-4C peptide was inserted into the HI loop of the fiber knob domain of the Ad.MCDIRESE1.71Hsp3 vector to increase the transduction efficiency of this vector to tumor cells. The Ad.MCDIRESE1.71Hsp3 vector replicates specifically in melanoma cells, and it has a melanoma-specific cytotoxic effect in the presence of 5-fluorocytosine in vitro and in vivo. Moreover, the in vivo killing of tumor cells associated with the overexpression of Hsp70 generated by the intratumoral injection of the Ad.MCDIRESE1.71Hsp3 vector into established subcutaneous tumors can lead to the suppression of tumor growth and potent melanoma-specific systemic immune responses.