Probiotics such as lactic acid bacteria (LAB) and prebiotics such as fiber are generally considered beneficial for health. These affect the microflora composition and fermentation metabolites and consequently contribute to local and systemic effects in humans. The beneficial effects of probiotics can be improved when combined with prebiotics. Here we investigated the effects of a mixed LAB supplement combined with dietary fiber on the population of LAB in the gut, as well as on serum cholesterol levels, fecal water content and microbial harmful enzyme activities. For animal studies, 0.2 mL of mixed LAB (Bifidobacterium longum SPM1205, Lactobacillus acidophilus, and SAFELAC isolated from Pediococcus pentosaceus) supplement (10(7) ∼ 10(8) colony forming units per day) was orally administered to male Sprague-Dawley rats every day for 2 weeks along with a diet containing 5% or 10% cellulose. The mixed LAB supplement combined with dietary cellulose significantly (p < 0.05) reduced serum total cholesterol and LDL levels. This combination also significantly (p < 0.05) increased the population of LAB and the fecal water content and significantly (p < 0.05) reduced microbial harmful enzyme (β-glucosidase, β-glucuronidase and tryptophanase) activities. These effects of LAB were particularly improved by its combination with 5% cellulose compared to the control (a diet without cellulose), and the 5% cellulose combination was more effective than the 10% cellulose combination. In conclusion, the incorporation of a fibrous diet such as cellulose with lactic acid bacteria improved the population of LAB, and daily consumption of this combination could reduce the serum cholesterol levels and activities of harmful enzymes such as β-glucosidase, β-glucuronidase, tryptophanase, urease in rats.