Regulation of innate immunity may be an effective means of cancer control. Delaying cancer onset is regarded as an important mode of action in cancer prevention. We have been investigating the chemopreventive mechanisms of Lactobacillus casei Shirota (LcS), a probiotic strain. In this study, we evaluated the effect of LcS on tumor onset and the involvement of natural killer (NK) cells using a 3-methylcholanthrene-induced carcinogenesis model. C3H/HeN mice were divided into three groups, according to treatment: vehicle-treated, treated with vehicle only; control, 3-methylcholanthrene treated; LcS, 3-methylcholanthrene and LcS treated. 3-Methylcholanthrene was injected intradermally at 7 weeks of age. LcS was mixed into the diet (0.05%, w/w), which the mice were fed from the day of 3-methylcholanthrene injection onward. Tumor incidence was observed weekly. Profiles of splenic NK cells, in vitro cytotoxicity and the proportion, in the early stage of carcinogenesis were analyzed at 5 weeks after the injection. The tumor suppressive effect of LcS was also evaluated in a beige mouse model that is genetically deficient in NK cells. LcS delayed tumor onset and reduced tumor incidence in the results with C3H/HeN mice (P< 0.05). More specifically, tumor incidence in the control group was 33% at 6 weeks after the injection and 83% at 11 weeks as opposed to 0 and 42%, respectively, in the LcS group. NK cell cytotoxicity was significantly higher than in the control group, and the number of NK cells also increased in the LcS group of C3H/HeN mice. However, LcS failed to suppress tumorigenesis in the beige mouse. These findings suggest that enhancement of the cytotoxicity of NK cells by LcS delays tumor onset.