Tumor antigen-specific cytotoxic T lymphocyte (CTL) is a promising agent for cancer therapy. Most solid tumors are characterized by increased interstitial fluid pressure (IFP) and dense collagen capsule, which form physical barriers to impede cancer treatment. However, it remains unclear how CTL-mediated anticancer response is affected at the presence of these obstacles. Using a microfluidic-based platform mimicking these obstacles, we investigated the migration characteristics and performance of anticancer response of CTLs targeting hepatic cancer cells via antigen-specific and allogeneic recognition. The device consisted of slit channels mimicking the narrow interstitial paths constrained by the fibrous capsule and increased IFP was simulated by applying hydrostatic pressure to the tumor center. We found that antigen-specificity of CTLs against the targeted cancer cells determined the cytotoxic efficacy of the CTLs but did not significantly affect the success rate in CTLs that attempted to infiltrate into the tumor center. When increased IFP was present in the tumor center, CTL recruitment to tumor peripheries was promoted but success of infiltration was hindered. Our results highlight the importance of incorporating the physical characteristics of tumor interstitum into the development of CTL-based cancer immunotherapy.