Animal science nutrition studies are increasingly focusing on finding solutions to reduce methane (CH4) emissions. In the present study, we evaluated the effect of reductive acetogenic bacteria [acetogen probiotics (AP)] and lauric acid (LA) on in vivo rumen fermentation and microbial populations in Hanwoo steers. Four cannulated Hanwoo steers (392 ± 14 kg) were analyzed in a 4 × 4 Latin square design and were placed in hood-type chambers. They were fed similar amounts of concentrate and rice straw within and experimental design as follows: control (Con; 40 g DM basal feed, nonaddition of AP or LA), T1 = LA (40 g DM basal feed mixed with 40 g LA), T2 = AP (40 g DM basal feed, fermented with AP), and T3 = LA + AP (40 g DM basal feed, fermented with AP and mixed with 40 g LA). The animals were acclimatized to the diet for 15 d, followed by 6 d of the experimental period. Rumen fluid samples for metabolite and molecular analyses were collected 6 h after the morning feeding, with 2-h collection intervals. The enteric CH4 production was monitored on the last 2 d of the experimental period. Concentrations of total volatile fatty acids increased with the increase in time after feeding. Acetate, propionate, and butyrate concentrations were observed to be higher in the treatments than in Con. The addition of LA and AP reduced CH4 emission compared with that of Con (P < 0.01). Nuclear magnetic resonance spectroscopy results revealed no correlation between the LA and Con groups, but AP showed a correlation with LA and Con. Reduction in the number of protozoa which was accompanied by a decrease, because methanogens live symbiotically with protozoa. Supplementation of AP or LA alone and in combination decreased (P < 0.05) the methanogen population, whereas supplementation of LA alone significantly increased (P < 0.05) Ruminococcus flavefaciens and slightly increased total fungi. Thus, dietary supplementation of LA and AP has inhibitory effects on CH4 production in Hanwoo cattle. If the effects of this method can be maintained, reductive acetogens could become an important part of strategies to lower ruminant CH4 emissions.