There is an unmet need for a biomarker of liver fat. We identified dimethylguanidino valeric acid (DMGV) as a circulating biomarker of liver fat. Here, we assess its two isoforms—symmetric (SDGV) and asymmetric (ADGV)—as biomarkers of steatosis. We determined plasma ADGV, SDGV, related metabolites, alanine aminotransferase (ALT), and the fatty liver index (FLI) in two cohorts and compared their diagnostic performance for liver fat detection. SDGV was the strongest predictor of moderate to severe steatosis. Changes in SDGV correlated with changes in liver fat % in a prospective cohort. In a murine model of fatty liver disease, protein levels and activity of alanine:glyoxylate aminotransferase 2 (AGXT2), which produces SDGV, were increased and coincided with elevation of SDGV concentrations. SDGV is a biomarker of liver fat and its increase in hepatic steatosis results from the upregulation of AGXT2 activity.