The objectives of this study were to determine hepatic patatin-like phospholipase domain-containing protein 3 (PNPLA3) protein abundance peripartum and the relationship with hepatic triglyceride (TG) content. Multiparous cows were blocked by expected calving date and randomly assigned to a control (n = 13) or fatty liver induction (FLI; n = 12) treatment. Control cows were fed ad libitum peripartum, while FLI cows were offered a 6 kg cracked corn top-dress prepartum and feed restricted to 80% ad libitum intake at +14 d relative to calving (DRTC) until blood β-hydroxybutyrate ≥3.0 mM. Liver biopsies were collected at −28, −14, +1, +14, +28, +42, +56 DRTC. Quantification of hepatic PNPLA3 via semiquantitative Western blot was normalized to total sample protein. Statistical analysis was performed using the GLIMMIX procedure (SAS 9.4). Hepatic TG %DM and PNPLA3 abundance were log₁₀ transformed. Models included fixed effects of treatment, DRTC, and treatment × DRTC, as well as random intercepts of cow, block, and repeated measures of cow across DRTC. The PNPLA3 model included the −28 DRTC abundance as a covariate. Hepatic PNPLA3 abundance was also regressed against hepatic TG. All FLI and 2 control cows had blood β-hydroxybutyrate ≥3.0 mM. Hepatic TG content did not differ between treatments (P = 0.50) but differed over time (P < 0.001). Prepartum hepatic TG was lower than postpartum; the postpartum maximum and nadir TG were at +14 and +56 DRTC (P < 0.05), respectively. No treatment difference was detected for hepatic PNPLA3 abundance (P = 0.31). A time effect was observed (P < 0.001) with increasing PNPLA3 from −14 to +56 DRTC. The greatest PNPLA3 abundance was observed during the postpartum decrease of hepatic TG. Hepatic PNPLA3 had an inverse relationship (β = −0.31; P = 0.03) when regressed against hepatic TG content. These results suggest hepatic PNPLA3 protein abundance was not altered by FLI but changed over time inversely compared with postpartum hepatic TG. Furthermore, regression analysis suggests that incremental increases in hepatic PNPLA3 may lessen TG accumulation peripartum.