Background: Anti-programmed death-1 (PD-1) monoclonal antibody, nivolumab, enhances antitumor activity by inhibiting the interaction of PD-1 and programmed death-1 ligand 1 (PD-L1) and has shown efficacy for advanced gastric cancer (AGC) in the salvage line. However, specific subsets of immune cells predominantly activated during the period of anti-PD-1 therapy for AGC have not been clarified. Methods: Peripheral blood mononuclear cells of 20 AGC patients treated with nivolumab were prospectively obtained before the initial and second administrations of nivolumab, and at the time of progressive disease (PD). The proportion of immune cell subsets were systematically analyzed by flow cytometry, including the expression of costimulatory and coinhibitory molecules such as T-cell immunoglobulin and mucin domain 3 (TIM-3), Lymphocyte-activation gene 3 (LAG-3), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T cell antigen-4 (CTLA-4), CD28, OX40, and inducible T cell costimulator (ICOS). Association between changes in the proportion of the subsets and therapeutic effect were analyzed. Results: Median progression free survival (PFS) of the whole patients was 51 days (95% CI 35–83). After a single course of nivolumab, patients showed a significant increase in activated effector memory and activated effector subsets of CD4+/CD8+ T cells (p = 0.018, 0.018, 0.032, 0.024). At the time of PD, proportions of myeloid dendritic cell, IgM memory B cell and Tfh-Th1/17 cell subsets decreased (p = 0.024, 0.013, 0.0039). On the other hand, LAG3 positive CD4+/CD8+ T cells, TIM-3 positive CD4+/CD8 T cells increased at the time of PD (p = 0.013, 0.032, 0.042, 0.042). Significant positive correlations were found between PFS and the proportion of LAG3 positive CD4+/CD8+ T cells (p = 0.0056, 0.0054), OX 40 positive CD4+/CD8+ T cells (p = 0.0034, 0.0006) prior to the initial nivolumab therapy. Conclusions: Nivolumab therapy enhances activation of effector memory and effector subsets of CD4+/CD8+ T cells. The expression level of LAG3 and OX40 on T cells might be correlated with efficacy of nivolumab therapy.