PNPLA3 (adiponutrin), a novel patatin‐like phospholipase domain‐containing enzyme, is expressed at high level in fat, but also in other tissues including liver. Polymorphisms in PNPLA3 have been linked to obesity and insulin sensitivity. Notably, a nonsynonymous variant rs738409(G) allele of the PNPLA3 gene was found to be strongly associated with both nonalcoholic and alcoholic fatty liver disease. We have generated Pnpla3^−/− mice by gene targeting. Loss of Pnpla3 has no effect on body weight or composition, adipose mass, or development, whether the mice were fed regular chow or high‐fat diet or bred into the genetic obese Lep^ob/ob background. Plasma and liver triglyceride content and plasma aspartate aminotransferase and alanine aminotransferase levels were not different between Pnpla3^+/+ and Pnpla3^−/− mice while they were on regular chow, fed three different fatty liver‐inducing diets, or after they were bred into Lep^ob/ob background. Hepatic Pnpla5 messenger RNA (mRNA) levels were similar in wild‐type and Pnpla3^−/− mice, although adipose Pnpla5 mRNA level was increased in Pnpla3^−/− mice. A high‐sucrose lipogenic diet stimulated hepatic Pnpla3 and Pnpla5 mRNA levels to a similar degree, but it did not affect adipose or liver triglyceride lipase (ATGL, known also as Pnpla2) mRNA in Pnpla3^+/+ and Pnpla3^−/− mice. Finally, Pnpla3^+/+ and Pnpla3^−/− mice displayed similar glucose tolerance and insulin tolerance tests while on regular chow or three different fatty liver–inducing diets. Conclusion: Loss of Pnpla3 does not cause fatty liver, liver enzyme elevation, or insulin resistance in mice. (HEPATOLOGY 2010)