This study was conducted to evaluate the effect of rumen origin lactate-utilizing bacteria (LUB) as one of the potential treatments on subacute ruminal acidosis (SARA) during in vitro challenge compared to buffering agents (NaHCO3, sea minerals, MgO) and direct-fed microorganism (yeast). We hypothesized that rumen LUB (RLUB) could be a potential treatment to treat ruminal acidosis. The supplementation level of other treatments was determined by referring to previous studies in the literature. The 108 CFU/g freeze-dried RLUB isolated from Hanwoo cattle were compared with 0.1% NaHCO3, 0.8% of MgO, 0.5% sea mineral and 0.4% yeast during in vitro SARA challenge. Rumen fluid collected from one cannulated Holstein and one Hanwoo steer fed by maize silage was mixed with 0.5g feed consisted of 0.05g forage and 0.45g concentrate. These mixtures were incubated in triplication for 3, 6, 12 and 24h. After 6h of incubation, along with MgO and sea minerals, RLUB treatment showed higher (p<0.05) ㏗ values than control with no significant differences in total volatile fatty acid concentration. However, in the same period, the propionate concentration and A:P ratio were higher in RLUB than in the other treatment (p<0.05), which might alter the fermentation pattern. On the other hand, the RLUB treatment produced a higher (p<0.05) ammonia-N concentration. Based on these results, we can conclude that RLUB might have the potential to alleviate SARA. Nonetheless, further study on its mechanism in SARA is required, especially with live animals.