3-Nitrooxypropanol (NOP) can reduce CH₄ production from the rumen of dairy cows and beef cattle, but the effect of NOP on the rumen microbiome is unknown. The current project aimed to evaluate the microbial profile changes in 4 studies with NOP fed to beef and dairy cows. Rumen contents were collected from 2 dairy (Study 1: Haisan et al., 2014; Study 2: unpublished data) and 2 beef (Study 3: Romero-Perez et al., 2014; Study 4: Romero-Perez et al., 2015) studies, and subjected to genomic DNA extraction. Bacterial, archaeal and protozoal rRNA fragments were amplified and subjected to pyrosequencing analysis using 454 Titanium FLX (Roche). The main bacteria phyla in the rumen were found to be affected by NOP treatments: in dairy, Firmicutes was greater in control than in NOP dairy cows for Study 1 (58 vs 55%, P = 0.03); while in beef, Bacteroidetes proportions varied among different NOP levels for Study 3 (from 31% for Control to 36% for NOP, P < 0.01) and among NOP treatment periods for Study 4 (P = 0.04). NOP also influenced other minor bacterial phyla, with the magnitude of impact differing among studies. The major methanogen genera were similar for all studies, and similar trends were found for both beef and dairy studies: Methanobrevibacter ruminanitum was greater (P < 0.05) in all 4 studies (57 vs 46%; 66 vs 51%; 52 vs 37%; 30 vs 20%) while Mbb. gottschalkii was less (P < 0.01) in cattle receiving NOP (Studies 2 to 4: 36 vs 20%; 59 vs 42%; 68 vs 54%). Mbb. smithii and Methanosphaera stadtmanae responded to NOP feeding in different ways depending upon the study. Entodiniomorphida predominated the protozoal community (>90%). The protozoa composition was not affected by NOP, and no clusters formed according to NOP dosage. In conclusion, NOP may reduce enteric CH₄ though changing rumen microbial community, but its effect of each phylotype was different, requiring further experimentation to understand its mode of action in altering the rumen microbiome.