Abstract Glycerin is an organic compound with an alcoholic function and can be esterified into fatty acids to form triglycerides. Due to the increasing availability of glycerin, studies that determine the best level of its inclusion in diets for ruminants are needed. This study evaluated the effects of glycerin supplementation on the proximate composition and fatty acid profile of the longissimus lumborum of heifers fed on Brachiaria brizantha pasture. Thirty-six heifers were distributed in a totally randomized design with four treatments (G0.0 = without glycerin, G4.6 = 4.6% glycerin, G9.3 = 9.3% glycerin and G14.3 = 14.3% glycerin). The addition of glycerin decreased the tetradecanoic and octadecanoic fatty acids but increased the pentadecanoic, heptadecanoic, heptadecenoic, eicosanoic, eicosatetraenoic and docosatetraenoic fatty acids. The saturated (SFA), monounsaturated (MUFA) and omega-3 (n-3) fatty acid concentrations were similar across the diets. However, the polyunsaturated (PUFA) concentrations and the PUFA:MUFA and n-6:n-3 ratios increased with the inclusion of glycerin in the diet. Glycerin levels up to 14.3% (corresponding to a substitution of 50.5% of corn for this byproduct as an energy source) did not alter the proximate composition of the meat but improved the fatty acid profile of the longissimus lumborum muscle and, consequently, increased the meat quality, potentially providing benefits for human health.