To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay–concentrate diet, was mixed with a N-free buffer–mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA), NH3-N and gas production were measured after a 24-h incubation period. Microbial biomass was estimated using adenine and guanine bases as internal markers, and ATP production was estimated stoichiometrically. Microbial growth efficiency (YATP) and gas efficiency (Egas) were estimated. Fermentation with casein as the sole N source was generally characterized by lower total VFA, NH3-N, total gas production and higher acetate : propionate (A : P) ratio and YATP than with urea. Results herein presented indicate that, under these in vitro conditions, caecal microbial population does in fact use urea N, but less efficiently than casein in terms of microbial growth.