Walnut kernel is often used to treat cholesterol gallstones (CGS) in China. Few studies have reported the effect of DWPE (defatted walnut powder extract) or walnut oil (WO) on anti-CGS and effect mechanism. In this studies, there were glansreginin A, ellagic acid, gallic acid, methyl gallate, and ethyl gallate in DWPE. Then, inhibitory effects of DWPE and WO on CGS were compared in C57BL/6 mice with a high-cholesterol lithogenic diet all the time or a lithogenic diet in initial weeks and a basal diet in subsequent weeks. Results showed that DWPE inhibited CGS with protective effect on liver damage. It markedly regulated lipid levels in the liver and the bile as well as downregulated the expression of proteins involved in cholesterol absorption, including ABCG5/8 and NPC1L1. Interestingly, DWPE had more inhibitory action on CGS than WO. Therefore, DWPE could reduce CGS effectively by downregulating the levels of ABCG5/8 and NPC1L1.