Calcium salts of long-chain fatty acids (CSFA) from linseed oil have the potential to reduce methane (CH₄) production from ruminants; however, there is little information on the effect of supplementary CSFA on rumen microbiome as well as CH₄ production. The aim of the present study was to evaluate the effects of supplementary CSFA on ruminal fermentation, digestibility, CH₄ production, and rumen microbiome <italic>in vitro</italic>. We compared five treatments: three CSFA concentrations—0% (CON), 2.25% (FAL) and 4.50% (FAH) on a dry matter (DM) basis—15 mM of fumarate (FUM), and 20 mg/kg DM of monensin (MON). The results showed that the proportions of propionate in FAL, FAH, FUM, and MON were increased, compared with CON (P &lt; 0.05). Although DM and neutral detergent fiber expressed exclusive of residual ash (NDFom) digestibility decreased in FAL and FAH compared to those in CON (P &lt; 0.05), DM digestibility-adjusted CH₄ production in FAL and FAH was reduced by 38.2% and 63.0%, respectively, compared with that in CON (P &lt; 0.05). The genera <italic>Ruminobacter</italic>, <italic>Succinivibri</italic>o, <italic>Succiniclasticum</italic>, <italic>Streptococcus</italic>, <italic>Selenomonas</italic>.<italic>1</italic>, and <italic>Megasphaera</italic>, which are related to propionate production, were increased (P &lt; 0.05), while <italic>Methanobrevibacter</italic> and protozoa counts, which are associated with CH₄ production, were decreased in FAH, compared with CON (P &lt; 0.05). The results suggested that the inclusion of CSFA significantly changed the rumen microbiome, leading to the acceleration of propionate production and the reduction of CH₄ production. In conclusion, although further in vivo study is needed to evaluate the reduction effect on rumen CH₄ production, CSFA may be a promising candidate for reduction of CH₄ emission from ruminants.