Background: Patients with many retrieved lymph nodes (LNs) have good outcomes in Stage II colon cancer. We previously reported that the longest diameter of retrieved LNs correlates with the number of LNs and can be used as a prognostic factor (Int J Colorectal Dis 2015). LNs consist mainly of T cells, B cells, NK cells, and histiocytes. We studied the relations of the number of NK cells and the T-cell and B-cell area ratios in longest-diameter LNs to the number of retrieved LNs and outcomes. Methods: The subjects were 320 patients with stage II colon cancer. One LN with the maximum long-axis diameter was selected, and was immunostained with CD56 as an NK-cell marker, CD3 as a T-cell marker, and CD20 as a B-cell marker. The number of CD56-positive cells were examined and the mean number per 0.093 mm² was calculated by 6 power fields. CD3 and CD20 positive area ratio was determined by image analysis. The relations were evaluated with the use of Spearman’s rank correlation coefficient. The cut-off values were calculated by ROC curve analysis. Results: The number of LNs was 14.8 ± 10.1 (mean ± SD). The number of CD56-positive cells was 10.7 ± 9.6. The CD3-positive area ratio was 0.39 ± 0.08. The CD20-positive area ratio was 0.42 ± 0.10. The correlation coefficients for the number of LNs with the number of CD56-positive cells, CD3-positive area ratio and CD20-positive area ratio were 0.30 (p < 0.001), -0.03 (p = 0.569) and 0.10 (p = 0.072), respectively. The number of LNs positively correlated with the number of CD56-positive cells. The median follow-up period was 118 months. Patients with 12 or more CD56-positive cells (n = 97) had a significantly better survival (HR, 0.64; 95%CI, 0.41-0.97; p = 0.040). Patients with a CD20-positive area ratio of 0.43 or greater (n = 160) had a significantly better survival (HR, 0.64; 95%CI, 0.44-0.92; p = 0.015). Multivariate analysis revealed the number of NK cells is an independent prognostic factor for relapse-free survival. Conclusions: A high number of NK cells in longest-diameter LN was associated with a higher number of retrieved LNs and better survival. NK cells in the longest LNs may reflect the host antitumor immune response in patients with Stage II colon cancer.