The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T‐cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8⁺ T cells using a tumor‐inoculated mouse model. The adoptive transfer of tumor‐specific CD8⁺ T cells cultured under glutamine‐restricted (dGln) conditions or CD8⁺ T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor‐inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD‐1 and increased Ki67 positivity among tumor‐infiltrating CD8⁺ T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8⁺ T‐cell exhaustion in vivo. Furthermore, the transferred CD8⁺ T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8⁺ T cells under Ctrl conditions. We found that the expression of a pro‐survival factor and memory T cell‐related transcription factors was significantly higher in CD8⁺ T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8⁺ T cells. The novel adoptive transfer of tumor‐specific CD8⁺ T cells cultured in glutamine‐restricted conditions may be a promising approach to improve the efficacy of cell‐based adoptive immunotherapy.