Significance A sequence variant (I148M) in PNPLA3 is a major genetic risk factor for nonalcoholic fatty liver disease. Previously, we showed that PNPLA3(148M) evades ubiquitylation-mediated degradation and accumulates to high levels on lipid droplets (LDs). Here we address how this accumulation is related to steatosis. We generated an active, ubiquitylation-resistant isoform that accumulated on LDs and increased hepatic triglyceride levels when expressed in livers of mice. Conversely, depletion of PNPLA3 resolved the excess hepatic fat accumulation associated with expression of PNPLA3(148M). Our results provide direct evidence that accumulation of PNPLA3 per se causes fatty liver, and that depletion of the protein is a potential strategy for therapeutic intervention.