Dietary probiotics supplementation exerts beneficial health effects. Since cigarette smoking reduces natural killer (NK) activity, we evaluated the effect of Lactobacillus casei Shirota (LcS) intake on NK cytotoxic activity in male smokers. The double-blind, placebo-controlled, randomised study was conducted on seventy-two healthy Italian blue-collar male smokers randomly divided for daily intake of LcS powder or placebo. Before and after 3 weeks of intake, peripheral blood mononuclear cells were isolated and NK activity and CD16+ cells' number were assessed. Daily LcS intake for 3 weeks significantly increased NK activity (P < 0·001). The increase in NK activity was paralleled by an increase in CD16+ cells (P < 0·001). Before intake, NK cytotoxic activity inversely correlated with the number of cigarettes smoked (R − 0·064). LcS intake prevented the smoke-dependent expected NK activity reduction. The analysis of the distribution of changes in smoke-adjusted NK activity demonstrated that the positive variations were significantly associated with LcS intake, while the negative variations were associated with placebo intake (median value of distributions of differences, 20·98 lytic unit (LU)/107 cells for LcS v. − 4·38 LU/107 cells for placebo, P = 0·039). In conclusion, 3 weeks of daily LcS intake in Italian male smokers was associated with a higher increase in cytotoxic activity and CD16+ cells' number in comparison to the placebo intake group.