In a Wistar rat model, prolonged supplementation of mustard seed (MS) to the diet significantly ameliorates the induction of colorectal carcinomas by 1,2-dimethylhydrazine (DMH). The expression of the splenocyte major histocompatibility complex class I (MHCI) was found significantly enhanced, whereas that of the major histocompatibility complex class II (MHCII) was significantly decreased. Compared to that of control animals, the proportion of spleenic B- and dendritic cells (DC) was amplified in the MS group. The expressions of MHCI, as well as that of MHCII, were increased in DC cells; whereas in B cells, MHCI expression was augmented but that of MHCII moderately decreased. The percentages of CD8+CD28+ and CD4+CD28+ cells were increased in the MS group, while the CD4+CD25+Foxp3+ subset was depressed. Plasma analysis showed that DMH-exposure induced amplified amounts of interleukin (IL)-4, IL-5, IL-10, and transforming growth factor-beta, whereas MS feeding counteracted this effect but enhanced IL-2, IL12p70, IL21, TNF-alpha, and interferon-gamma. In the SW480 colon adenocarcinoma cell-line, the cytotoxicity of spleenic T-cells from MS-fed animals was significantly increased. In the DMH-exposed rats, the expression of perforin in the spleenic T-cells was dramatically decreased, whereas MS abolished this depression. In summary, dietary MS suppresses DMH-induced immuno-imbalance as well as colon carcinogenesis in rats.