Butyrate has been proposed as an antineoplastic agent, leading to the inhibition of tumorigenesis. The purpose of this study was to examine butyrate, supplied as tributyrin (Tbn) or as a natural component of anhydrous milk fat (AMF), on the development of nitrosomethylurea-induced mammary tumors in female Sprague-Dawley rats. Diets were 1) semipurified rodent diet (AIN-93) with high fat [20% sunflower seed oil (SSO), control], 2) SSO diet with Tbn added at 1%, 3) SSO diet with Tbn added at 3%, and 4) 19% AMF with 1% SSO diet, which contained butyrate equivalent to the 1% Tbn diet. These diets were fed ad libitum from weaning at 21 days of age, and at 24 days of age each rat was injected with nitrosomethylurea (50 mg/kg body wt i.p.). At any one period, there was a relative risk increase of 88% (p < 0.05) that rats in the SSO diet group would develop a mammary tumor compared with those in the AMF diet group. The addition of 1% and 3% Tbn to SSO diets reduced the tumor incidence by 20% and 52%, respectively, in comparison to SSO alone (p < 0.05). There was a linear inverse relationship between Tbn concentration and rats developing a tumor. From 89 days to the end of the experiment, rats fed the diet containing 3% Tbn showed a significantly lower multiplicity of palpable tumors (50% less at Day 118, p < 0.05) than SSO-fed rats. These results indicate that although the AMF diet was effective, particularly early in reducing mammary tumorigenesis, the 3% Tbn diet produced a sustained reduction of tumor multiplicity relative to the control (SSO) group. An inhibitory influence of butyrate on mammary tumorigenesis against a background of high polyunsaturated fat diet has been demonstrated in this animal model of breast cancer.