Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a membrane bound lipase that has been associated with the accumulation, and potentially with the subsequent depletion, of liver lipids in dairy transition cows. Hepatic PNPLA3 abundance is increased postcalving during remobilization of liver lipids, and is correlated with nonesterified fatty acids (NEFA) concentrations. Fatty acids are known regulators of hepatic gene and protein abundance. Therefore, the objective of this study was to determine if hepatic PNPLA3 abundance is responsive to incremental changes in concentration of fatty acids in primary hepatocytes. Primary hepatocytes isolated from 4 Holstein calves were maintained as monolayer cultures for 24 h before treatment. Treatments consisted of a NEFA free control, palmitic acid (C16:0), oleic acid (C18:1n-6), α-linolenic acid (C18:3n-3), and a fatty acid cocktail with a profile of fatty acids reflective of NEFA at parturition. The fatty acid cocktail consisted of 3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1n6, 8% C18:2 n-6, and 8% C18:3n-3. Abundance of PNPLA3 was determined by Western blot analysis and normalized to total lane protein. For analysis, PNPLA3 was expressed relative to a fatty acid free control, and transformed as log(relative abundance +1) because data were not normally distributed. Data were analyzed for main effects of treatment and concentration, and linear and quadratic contrasts of concentration using PROC MIXED in SAS 9.4. Addition of any fatty acids decreased (P < 0.05) PNPLA3 compared with fatty acid free control. Increasing fatty acid concentrations linearly decreased (P < 0.05) PNPLA3 abundance. Fatty acid treatment had an effect (P < 0.05) on PNPLA3 abundance with the fatty acid cocktail tending to result in greater PNPLA3 abundance than palmitic acid. These data indicate that PNPLA3 abundance may be regulated by fatty acids. This could suggest that changing fatty acid concentrations during the transition period could play a role in regulating PNPLA3 abundance during this time.