DNA vaccines provide an attractive technology against cancer because of their safety record in humans and ease of construction, testing and manufacture. In this study, several DNA fragments encoding multiple cytotoxic T lymphocyte (CTL) and T helper cell epitopes were selected from human prostate‐specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate‐specific antigen (hPSA). These DNA fragments were ligated together to form a novel fusion gene, termed the 3P gene. The secondary lymphoid tissue chemokine (SLC), 3P and human immunoglobulin G Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC‐3P‐Fc. After vaccination, the DNA is taken up by cells that produce and secrete the SLC‐3P‐Fc fusion proteins, termed chemotactic antigen (chemo‐antigen). The secreted chemo‐antigens, in addition to promoting the co‐localization of naive, non‐polarized memory T cells and dendritic cells, are efficiently captured and processed by dendritic cells via receptor‐mediated endocytosis and then cross‐presented to both major histocompatibility complex class I and class II in a cognate manner. The results of this study demonstrate that vaccination with pSLC‐3P‐Fc by gene gun inoculation induced a strong antitumour response in a mouse tumour model, which significantly inhibited tumour growth and prolonged the survival time of the tumour‐bearing mice. In vitro, the secreted SLC‐3P‐Fc fusion protein can attract lymphocytes from human peripheral blood mononuclear cells (PBMC); when human lymphocytes were stimulated by pSLC‐3P‐Fc‐transfected autologous PBMC, CTLs were induced which could specifically kill hPSM‐, hPAP‐, or hPSA‐expressing tumour cells. These observations provide a new vaccine strategy for cancer therapy through promoting the co‐localization of lymphocytes and the concomitant enhancement of antigen‐specific CD4⁺ helper and CD8⁺ cytotoxic T‐cell responses against tumour.