Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a membrane bound triglyceride lipase that contributes to lipid metabolism in the liver and is responsive to feed restriction. PNPLA3 mRNA expression is dynamic in dairy cows with lower expression during negative energy balance. The objective of this study was to characterize PNPLA3 protein abundance in transition cows subjected to fatty liver induction. Multiparous cows were blocked by expected calving date and randomly assigned to a control (n = 3) ad libitum intake group, or a fatty liver induction (n = 6) group that was overfed during the dry period, and feed restricted (80% of dry matter intake) at +14 d relative to calving (DRTC) until onset of clinical ketosis. Liver biopsies were taken in the prepartum (−28, −14 DRTC), lipid accumulation (+1, +14, +28 DRTC), and recovery (+42, +56 DRTC) periods and at the time of clinical ketosis. Liver PNPLA3 protein abundance was determined through Western blot analysis and normalized to total protein. Data were analyzed using PROC MIXED of SAS 9.3. Abundance of PNPLA3 was analyzed with main effects of treatment, period, and treatment x period, and random effect of cow. Protein abundance was also analyzed by PROC MIXED and PROC CORR based on total lipid accumulation diagnosed as high (>15%, dry matter) or low (<15%, dry matter). All fatty liver induction cows became clinically ketotic and developed fatty liver. Abundance of PNPLA3 was greater (P = 0.02) during the recovery period compared with the accumulation period (1.12 vs. 0.79 ± 0.09, arbitrary units). Cows with high liver lipids had decreased (P < 0.01) PNPLA3 abundance compared with cows with low liver lipids (0.69 vs. 1.0 ± 0.08, arbitrary units). There was a negative correlation (P < 0.01; r = −0.427) between liver lipid concentration and PNPLA3 abundance. These data indicate that regulation of PNPLA3 may influence liver lipid accumulation and potential recovery from fatty liver disease.