Glycerol is rapidly fermented to propionate in the rumen. Due to the inverse relationship between propionate production and enteric CH4 emissions, glycerol may reduce rumen CH4 production. However, little data have been reported to substantiate this hypothesis. Our objectives were to determine ruminal fermentation rate of glycerol and effects of glycerol on in vitro fermentation and CH4 production. Glycerol (0.5g), alfalfa hay (0.5g), corn grain (0.5g), alfalfa hay (0.5g)+glycerol (0.1g) and corn grain (0.5g)+glycerol (0.1g) were anaerobically incubated in vitro using strained rumen fluid for 0, 3, 6, 12, 24 and 48h. At each incubation time, total gas and CH4 production, pH, ammonia and volatile fatty acid concentrations (VFA) were measured. By fitting an exponential model with a single lag, a fractional gas production rate of 0.051/h with a lag of 7.9h for glycerol, which was a slower rate and a longer lag than for the other substrates (P<0.01), was obtained. Theoretical maximum gas production from glycerol was 243ml/g. Fermentation of glycerol produced VFA with a high proportion of propionate, and had the lowest acetate:propionate ratio among treatments at 24h of incubation (P<0.01). Adding glycerol decreased total gas and CH4 production (P<0.01), but not VFA production at 24h. Results suggest that adding glycerol to a diet may increase efficiency of energy use of feeds by ruminants. Based on the amount of digestible energy (DE) in each treatment, glycerol supplementation decreased CH4 production/unit DE (P<0.01). Fermentation of glycerol is relatively slow, but the rapid increase in gas production from glycerol after a lag time indicates that fermentation kinetics of glycerol may be altered by adaptation of the rumen microbial population to glycerol. On the basis of the DE level of our substrates, glycerol supplementation may decrease CH4 production and increase efficiency of dietary energy use in ruminant diets.This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.