It is widely reported how betaine addition regulates lipid metabolism but how betaine affects cholesterol metabolism is still unknown. This study aimed to investigate the role of betaine in hepatic cholesterol metabolism of Sprague-Dawley rats. Rats were randomly allocated to four groups and fed with a basal diet or a high-fat diet with or without 1% betaine. The experiment lasted 28 days. The results showed that dietary betaine supplementation reduced the feed intake of rats with final weight unchanged. Serum low-density-lipoprotein cholesterol was increased with the high-fat diet. The high-fat diet promoted cholesterol synthesis and excretion by enhancing the HMG-CoA reductase and ABCG5/G8, respectively, which lead to a balance of hepatic cholesterol. Rats in betaine groups showed a higher level of hepatic total cholesterol. Dietary betaine addition enhanced cholesterol synthesis as well as conversion of bile acid from cholesterol by increasing the levels of HMGCR and CYP7A1. The high-fat diet decreased the level of bile salt export pump, while dietary betaine addition inhibited this decrease and promoted bile acid efflux and increased total bile acid levels in the intestine. In summary, dietary betaine addition promoted hepatic cholesterol metabolism, including cholesterol synthesis, conversion of bile acids, and bile acid export.