Fatty liver syndrome is a prevalent metabolic disorder in peripartum dairy cows that unfavorably impacts lactation performance and health. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) is a lipase that plays a central role in human non-alcoholic fatty liver disease etiology but has received limited attention in bovine fatty liver research. Thus, we investigated the relationship between tissue PNPLA3 expression and liver triglyceride accumulation in vivo via a ketosis induction protocol in multiparous dairy cows peripartum, as well as in vitro via small interfering RNA knockdown of PNPLA3 mRNA expression in bovine primary hepatocytes. Results demonstrated a negative association (P = 0.04) between liver PNPLA3 protein abundance and liver triglyceride content in peripartum dairy cows, while adipose PNPLA3 protein abundance was not associated with liver triglyceride content or blood fatty acid concentration. Knockdown of PNPLA3 mRNA resulted in reduced PNPLA3 protein abundance (P < 0.01) and greater liver triglyceride content (P < 0.01). Together, these results suggest greater liver PNPLA3 protein abundance may directly limit liver triglyceride accumulation peripartum, potentially preventing bovine fatty liver or accelerating recovery from fatty liver syndrome.