Adoptive cellular transfer has been employed for cancer immunotherapy, including patients with gastric cancer. However, little is known about the distribution of effector cells after their injection via different pathways. In this study, we used human gastric cancer cells (BGC823) tagged with enhanced green fluorescent protein (EGPF) to establish a subcutaneous gastric cancer model in nude mice. Cytokine-induced killer (CIK) cells and cytotoxic T lymphocytes (CTLs) were generated from human peripheral blood and labeled with red fluorescent PKH26. A portion of CIK cells was armed with CEA/CD3-bispecific single-chain antibody. When CIK cells were injected into nude mice with established subcutaneous gastric cancer via peritumoral (p.t.), intravenous (i.v.) and intraperitoneal (i.p.) infusion respectively, the distribution of cells was observed using a live fluorescence imaging system. We found that only a very small number of CIK cells could travel to the tumor site after i.p. or i.v. infusion, and they inhibited subcutaneous tumor growth in vivo only immediately following injection. In contrast, p.t. injection resulted in a significantly higher accumulation of CIK cells at the tumor site for 48 hours and mediated the greatest tumor inhibition compared with the other two injection methods. In addition, we compared the antitumor activity of CIK, CEA/CD3-bscAb-CIK and CTL cells in vitro and in vivo after p.t. injection. Among the three types of immune cells, CTLs demonstrated the strongest antitumor activity both in vitro and in vivo. CEA/CD3-bispecific single chain antibody could effectively link T lymphocytes and tumor cells expressing CEA, and resulted in significantly higher accumulation of CIK cells at the tumor site compared with the parental CIK cells. This study indicates that peritumoral injection of immune effector cells by minimally invasive surgical procedures represents an effective delivery method of adoptive cellular immunotherapy. Tumor-specific immune cells, such as CTLs, are a better choice of effector cells than CIKs in cellular immunotherapy. Furthermore, CD3+ immune cells armed with the CEA/CD3-bispecific single chain antibody could more effectively travel to and accumulate at the site of tumors expressing CEA, such as gastric cancer.