We recently reported that fermentable non-digestible carbohydrates including fructo-oligosaccharides (FOS) commonly elevate colonic alkaline phosphatase (ALP) activity and the expression of IAP-I, an ALP gene, in rats fed a high-fat (HF) diet, and also elevate gut mucins and modulate gut microbiota. This study aims to investigate whether dietary fat types influence the effect of FOS on colonic ALP activity and the luminal environment in HF-fed rats. Male Sprague-Dawley rats were fed a diet containing 30% soybean oil, corn oil, olive oil or lard with or without 4% FOS for 2 weeks. Colon ALP activity, gene expression, and gut luminal variables including mucins and microbiota were measured. In the lard diet groups, dietary FOS significantly elevated colonic ALP activity and the expression of IAP-I. The elevating effect of FOS on colonic ALP activity was also observed in the olive oil diet groups, although here the IAP-I expression was not changed. However, the soybean oil and corn oil diet groups did not exhibit the elevating effect of FOS on colon ALP. Fecal ALP and mucins were significantly elevated by dietary FOS regardless of dietary fat types, and the effect of FOS was prominent in the lard diet groups. The number of Lactobacillus spp. observed in fecal matter was significantly increased by dietary FOS in the lard and olive oil diet groups, but not in the soybean oil and corn oil diets groups. This study suggests that dietary fat types may change the effect of FOS on the colonic luminal environment including the ALP activity in rats fed a high-fat diet.