Adoptive T cell immunotherapy with cytokine-induced killer cells (CIKs) has been demonstrated to prolong the survival of patients with advanced non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate whether the expansion of effector T cells and the decrease of regulatory T cells (Tregs) that occurred during the generation of DC-CIKs were associated with improved clinical outcome in patients who received treatment. CIKs were generated over a 28-day period from the peripheral blood apheresis product of 163 patients with advanced cancer (including 30 with NSCLC). CIKs were also generated from an additional cohort of 65 patients with NSCLC over a 15-day period. The progression-free survival (PFS) and overall survival (OS) time of patients treated with CIKs was determined by reviewing the patients' medical records. The number of CIKs gradually increased during the culture period and peaked at day 15, followed by a slight decline until day 28. Similarly, the percentages of T cell subtypes associated with anti-tumor activity (CD3, CD3CD4, CD3CD8 and CD8CD28) peaked at day 15. Although the percentage of CD4CD25CD127 Tregs increased by day 7, a decrease was subsequently observed. Among the 95 patients with NSCLC, those with a post/pre-culture ratio of CD8CD28 T lymphocytes >2.2 had significantly better PFS and OS compared with those with ratios ≤2.2. Those with a post/pre-culture CD4CD25CD127 Treg ratio ≤0.6 had significantly better OS and PFS compared with those with ratios >0.6. The peak expansion of CIKs from peripheral blood mononuclear cells occurred at day 15 of culture. PFS and OS were associated with post/pre-culture CD8CD28 T lymphocyte ratio >2.2 and post/pre-culture CD4CD25CD127 Treg ratio <0.6 in the CIKs of patients with advanced NSCLC treated with adoptive T cell immunotherapy. Further efforts are underway to optimize the DC-CIK infusion for cancer immunotherapy.