The study evaluated the effect of Entodinium caudatum on the prevention of subacute ruminal acidosis (SARA) in vitro. Different proportions of wheat and corn [100% wheat (W); 75% wheat and 25% corn (W75); 50% wheat and 50% corn (WC); 75% corn and 25% wheat (C75); and 100% corn (C)] were used to create an in vitro acidotic environment. The activity of E. caudatum was determined by adding defaunated rumen fluid and protozoan monocultures to the substrates. The effect of E. caudatum monoculture on pH was insignificant, while a significant influence of grain type was observed on ammonia (NH3-N) formation. E. caudatum inoculation decreased lactic acid concentration throughout the incubation period and shortened the fermentation start time (lag time). The specific fermentation rate (SFR), which increased with the wheat ratio, was reduced by E. caudatum culture. The total gas production varied depending on the substrate and was increased by E. caudatum. Protozoan monoculture decreased propionic acid levels, while it increased methane production. As a result, E. caudatum stimulated earlier fermentation, but decreased lactic acid production by reducing SFR. It is believed that the instantenuous phagocytosis of E. caudatum (iodophilic storage) to digest starch particles prevents rapid bacterial fermentation in the rumen fluid, and thus limit lactic acid production. The results of this work may support future in vitro studies requiring E. caudatum monoculture, as well as in vivo studies investigating the effect of E. caudatum on the inhibition of SARA formation.