This study was carried out to investigate effects of type and level of octadeca carbon fatty acids (C 18 -fatty acids) on rumen fermentation, methane emission and populations of ruminal microbes using an in vitro gas production technique. Four types of C 18 -fatty acids, stearic, oleic, linoleic and linolenic acids, were added at levels of 0, 35 and 70g/kg of substrate dry matter, respectively. Methane emission was determined by gas chromatography. Total deoxyribonucleic acid (DNA) was extracted from ruminal microbes, and populations of rumen microbes were determined by a real-time quantitative polymerase chain reaction. Populations of total rumen methanogens, protozoa, fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes were expressed as a proportion of total rumen bacterial 16S ribosomal deoxyribonucleic acid (rDNA). The 24h gas production and methane emission were decreased by type, level and their interaction (P<0.001). Compared to the control, C 18 -fatty acids had little effect on pH, ammonia N and total volatile fatty acids. However, the fermentation patterns were changed, reflected by lower proportion of acetate and higher propionate with increasing levels and unsaturation of C 18 -fatty acids (P<0.001). Methanogens and protozoa populations relative to total bacterial 16S rDNA were decreased by linoleic and linolenic acids, with the linolenic acid addition being most efficient. However, addition of these unsaturated C 18 -fatty acids also inhibited growth of fibrolytic microbes including fungi, F. succinogenes and R. flavefaciens. From the present study, it is inferred that there is an effect of unsaturated C 18 -fatty acids in suppressing methanogenesis, probably mediated by direct action against the rumen microbes involved in methane formation.