Abstract: In order to investigate the effects of tobacco seed oil on serum cholesterol level and expressions of regulatory genes related to cholesterol metabolism in livers of rats, the levels of total cholesterol (TC), triacylglycerol (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) in serum were determined by using rats fed with a high level cholesterol diet as animal models. The rats were randomly divided into control groups (blank control group, model control group, fish oil control group) and experimental groups (three groups fed with tobacco seed oil at low, middle and high doses, separately). The expressions of regulatory genes related to cholesterol metabolism in livers of rats were measured with real-time PCR method. Liver tissues were sliced and stained to observe the steatosis of hepatic cells of rats. The results showed that:1) Tobacco seed oil effectively reduced the levels of TC and LDL-C in serum and atherogenic index of rats, improved hepatic steatosis. 2) Tobacco seed oil down-regulated the expression of HMGCR gene and up-regulated the expression of LDLR gene in livers of rats with hypercholesterolemia. Therefore, tobacco seed oil has a significant effect of lowering total cholesterol, the mechanism might be actioned by regulating the expressions of genes related to cholesterol metabolism in livers.