The effect of crude glycerin (CG) - 80% of glycerol - inclusion as a substitute to corn grain in supplements on rumen fluid protozoa numbers, and relative proportion of cellulolytic bacteria and methanogenic archaea of rumen-cannulated Nellore steers (n = 10; BW = 408.8 ± 38.5 kg) on low quality pasture, in the dry season was studied. Treatments were constituted by 5 levels of CG in the supplement: 0, 7, 14, 21 and 28% DM of CG. Animals were supplemented, daily at 1000 h in a ratio of 700 g/100 kg of BW. Supplement consisted of corn grain, soybean meal, urea, gluten meal and mineral mix. Bacteria and protozoa samples were collected on the d 11 of each experimental period, 3 h after supplementation, in the solid and liquid extracts in different parts of the rumen. Real-time PCR was used to quantify microbial population. Methanogens and cellulolytic bacteria were expressed as a proportion of total rumen bacterial 16S rDNA. Ciliate protozoa species were identified and quantified (#/mL) in a Sedgewick-Rafter chamber. Data of ciliated protozoa were log10-transformed and analyzed in a replicated Latin square design using the MIXED procedure of SAS. Bacterial proportions were analyzed using the software R, with data compared between treatments (with or without 28% DM of CG in the supplement) using the Wilcoxon test, considered significant effects at P < 0.05. The inclusion of CG in supplement did not affect (P > 0.05) rumen protozoa number (6.52, 4.46, 3.02, 2.95, 1.73, 2.16 and 1.67 for the genera Entodinium, Dasytricha, Isotricha, Eremoplastron, Eudiplodinium, Elytroplastron, and Polyplastron). The inclusion of CG had no effect in the rumen relative proportion of Ruminococcus albus (P = 0.237), Ruminococcus flavefaciens (P = 0.129), and methanogens (P = 0.151) with mean values of 0.0046, 0.0044, and 0.0169. However, increased (P = 0.003) Fibrobacter succinogeneses, with mean values of 0.0033 and 0.0296 for 0% DM and 28% DM of CG in the supplements, respectively. Inclusion up to 28% DM of CG in the supplement did not interfere negatively on rumen microbial profile, increased relative proportion of F. succinogenes of Nellore steers in low quality pasture.