The present study compared the hepatic fatty acid composition of cows that were fed a high energy diet during the dry period to induce fatty liver after parturition with that of control cows. Treated cows had higher concentrations of plasma nonesterified fatty acids as a result of greater lipolysis after parturition than did control cows; consequently, the treated cows accumulated greater amounts of triacylglycerols in the liver. Before parturition, treated cows had lower percentages of oleic acid and higher percentages of linoleic acid than did control cows, but percentages of other fatty acids were similar for both groups. After parturition, percentages of each fatty acid were changed substantially, particularly the four major fatty acids (palmitic, stearic, oleic, and linoleic acids). For treated cows, the percentages of palmitic and oleic acids were higher at 0.5 wk after parturition than at 1 wk before parturition; percentages of stearic and linoleic acids decreased. Unlike treated cows, the percentages of both oleic and linoleic acids in the control cows did not change during that time. Moreover, we found that when lipolysis decreased, as indicated by lower plasma nonesterified fatty acid concentrations, the percentages of each fatty acid gradually rebounded toward the concentrations measured before parturition; this observation indicates that the shift in hepatic fatty acid composition is influenced by lipolysis. The increased lipolysis after parturition led to a vast increase in the hepatic triacyglycerol concentration and to a shift in hepatic fatty acid composition.