The current study aimed to investigate the antitumor effects and potent mechanism of cytokine-induced killer (CIK) cells combined with irreversible electroporation (IRE) via an in vitro function assay in an in vivo Panc02 cell–bearing mouse model. We established an in vitro culture assay for CIK cells and determined the proportions of different peripheral lymphocytes. The antitumor effect of the combination of IRE and CIK cells in a Panc02 pancreatic-cancer (PC) subcutaneous-xenograft model was investigated; tumor size and mouse survival rates were recorded. We used flow cytometry (FCM) to analyze the proportion of intratumoral lymphocytes, the expression of chemokine receptors, and the proliferative activity of CIK cells. The proportion of cells that were positive for clusters of differentiation 3 and 8 (CD3+CD8+) and the proportion of CD3+CD56+ cells were both significantly increased after 21 days of in vitro culture. Combined IRE/CIK cell treatment significantly inhibited tumor growth and increased the survival rate of Panc02 cell–bearing mice. Furthermore, infiltration of lymphocytes into tumor tissue was significantly increased by this combination therapy compared with the untreated group or monotherapy group. In addition, IRE significantly enhanced the expression of chemokine receptors elicited by, and the proliferative activity of, CIK cells. In conclusion, IRE combined with CIK cells showed superior antitumor efficacy in a PC subcutaneous-xenograft model, which we attributed to the promotion of lymphocytic infiltration, as well as to upregulation of chemokine receptor expression and of regulators of CIK cell proliferation.