The regional standard for tempeh established by the Codex Alimentarius defines the use of Rhizopus oligosporus, R. oryzae, and/or R. stolonifer as soybean tempeh starters. However, comparative studies on the functions of tempeh prepared with these Rhizopus species are scarce. In the present study, we examined the effects of dietary tempeh prepared with these three Rhizopus species using rats fed with a high-fat diet. Compared to the control diet, consumption of tempeh prepared with R. stolonifer significantly suppressed serum levels of aspartate transaminase, total bilirubin, and ammonium (indices of liver function). However, less or no suppression was observed with tempeh prepared with R. oligosporus or R. oryzae. Serum levels of triglyceride, total cholesterol, HDL cholesterol, and glucose were unaffected. Liver levels of free cholesterol, a parameter relating to liver injury, were significantly decreased by the three types of the tempeh examined; however, there was no difference in the free cholesterol levels among the tempeh groups. We conclude that the ingestion of tempeh prepared with R. stolonifer might have beneficial effects pertaining to the liver function in rats fed with high-fat diets.