An experiment was conducted with laying hens to determine the AMEn value of crude glycerol, a coproduct of biodiesel production. Crude glycerol (87% glycerol, 9% water, 0.03% methanol, 1.26% Na, and 3,625 kcal/kg of gross energy) was obtained from a commercial biodiesel production facility (Ag Processing Inc., Sergeant Bluff, IA). A total of forty-eight 40-wk-old laying hens (Hy-Line W-36) were placed in metabolic cages (2 hens/ cage) and given free access to the experimental diets. A corn and soybean meal-based basal diet (18% CP, 2,875 kcal/kg of AMEn, 4.51% Ca, 0.51% nonphytate P) was formulated with 15% glucose·H2O and 1% Celite. Four dietary treatments were created by substituting 0, 5, 10, or 15% crude glycerol for glucose·H2O (3,640 kcal/kg of AMEn). After 7 d of dietary adaptation, excreta were collected twice daily for 3 d, freeze-dried, and analyzed for contents of DM, Kjeldahl N, acid-insoluble ash, and gross energy. Egg production was recorded daily, and eggs were collected on d 7 and 8 of the experiment for calculation of egg mass (egg production × egg weight). Feed consumption was measured over the 10-d experimental period. Egg-production data were analyzed by ANOVA with 4 treatments and 6 replications in a completely randomized experimental design. The AMEn value of crude glycerol was estimated as the slope of the linear relationship between the inclusion rate of dietary crude glycerol and the glucose-corrected AMEn value of the experimental diets. No significant treatment effects (P > 0.1) were apparent for egg-production rate (93.0%), egg weight (56.1 g), egg mass (52.2 g/d), or feed consumption (104 g/d). Linear regression analysis (P < 0.001, r2 = 0.92, n = 24) revealed that the AMEn value of the crude glycerol used in this study was 3,805 ± 238 kcal/kg (mean ± SEM; as-is basis) for laying hens.