Alfonso-Ávila, Á. R., Charbonneau, E., Lafrenière, C. and Berthiaume, R. 2015. Effect of glycerol in combination with alfalfa on in vitro gas production and microbial protein synthesis. Can. J. Anim. Sci. 95: 577-588. This study sought to determine the effect of added glycerol on microbial protein synthesis, ruminal degradation and utilization of alfalfa at different concentrations of nonstructural carbohydrates (NSC), using in vitro gas production. The 2×3 factorial plus one treatment consisted of oven-dried alfalfa with two NSC levels [high: 17.9 (HNSC) or low: 7.4% dry matter (DM) (LNSC)] and three glycerol treatments [control without glycerol, 15% crude glycerol (CG) and 15% pure glycerol (PG)], the additional treatment was LNSC exogenous sugars (LNSC ES: LNSC with 5% sucrose 5% starch). Five pre-planned contrasts were evaluated from the seven treatments: (1) HNSC vs. LNSC alfalfa; (2) with glycerol vs. without; (3) interaction of alfalfa and glycerol; (4) CG vs. PG, and; (5) LNSC ES vs. HNSC. Gas production over 24 h was higher for HNSC than LNSC (202 vs. 179 mL g^-1 DM) and with glycerol than without glycerol (202.2 vs. 168 mL g^-1 DM). A decrease in the acetate:propionate ratio was observed for HNSC compared with LNSC (2.87 vs. 3.27) and for the addition of glycerol vs. no glycerol (2.78 vs. 3.65). Reduced microbial mass (185.5 vs. 240.5 mg g^-1 DM) was observed for CG compared with PG. The LNSC ES treatment had lower microbial protein synthesis and propionic acid production in relation to HNSC. No significant interaction was observed between the effect of NSC content of alfalfa and glycerol utilization. When effects were studied separately, results indicate that increasing NSC in alfalfa stimulates the synthesis of microbial protein. Also, the addition of glycerol promotes the synthesis of glucose precursors. Finally, the type of glycerol has an impact on results obtained suggesting caution when extrapolating results for PG to CG.