USP21, a member of the ubiquitin protease family, plays a vital role in various biological functions. However, the effects of USP21 dysfunction have yet to be fully understood. Here, we generated Usp21 knockout (KO) mice and conducted microarray to identify genes that Usp21 regulates. Its results raised several genes based on their biological characteristics, and RT-qPCR confirmed their expression. KO mice exhibited significant elevations in the expression of the genes Fabp7 and Nlrc5, which play a role in lipid metabolisms, compared to wild-type mice. Blood tests showed no impairment of liver function but did reveal elevated levels of total cholesterol and free fatty acids in KO mice compared to wild-type mice. These data suggest that Usp21 may play roles in lipid metabolism associated with Fabp7 and Nlrc5. To clarify the involvement of USP21 in human hypercholesterolemia, we examined single nucleotide polymorphisms (SNPs) around USP21 in non-hypercholesterolemic and hypercholesterolemic outpatients. We found that the rs11421 SNP downstream of USP21 was associated with hypercholesterolemia. These data suggest that USP21 plays a role in mice and human lipid metabolism and that its polymorphism may be a diagnostic marker for human hypercholesterolemia.