The aim of this study was to evaluate the effects of different sources of nitrogenous compounds on the in vitro utilisation of neutral detergent fibre from buffel grass in advanced phenological stage, the experiment consisted of testing five levels of substitution of urea for casein: 0, 25, 50, 75 and 100%. The effects of the substitution levels were evaluated by in vitro incubation at different times: 0, 3, 6, 9, 12, 24, 36, 48, 72 and 96 h. The degradation rate of potentially degradable NDF increased up to the replacement level of 50%, but declined by 6.53 and 13.57% in the treatments with 75 and 100% substitution of urea for casein, respectively, as compared with the treatment without substitution. Discrete lag time was reduced by 1.31 h in the treatment with 50% substitution and by 2.7 h at 100% substitution, as compared with 0% substitution. The substitution of up to 50% non-protein nitrogen for true protein increased microbial growth efficiency by 16.1% as compared with the treatment without substitution. Acetate and propionate concentrations were not affected by the substitution of urea for casein. The use of 50% non-protein nitrogen and 50% true protein as nitrogen sources for rumen microorganisms favour microbial growth and optimise the degradation of neutral detergent fibre from low-protein buffel grass. © 2021 Friends Science Publishers