The objective was to evaluate the effect of feed inoculation with effective microorganisms (EM®) (mainly containing Lactobacillus spp.,Rhodopseudomona palustrisand Saccharomyces cerevisiae) on rumen fermentation using in vitrogas production technique. We hypothesized that increasing doses and allowing exposure of EM® for up to 48 hours, would improve digestibility and rumen fermentation. The experimental design was a 4×4 completely randomized block design including 4 EM® levels [(0(EM0), 0.5(EM0.5), 1.0 (EM1) and 1.5 (EM1.5) mL EM® / kg DM] and 4 preincubation times [0 (T0), 12(T12), 24(T24), 48 (T48) h], with four repetitions per treatment. Treatments were evaluated using 100ml glass bottles with 0.5g of the diet (20% corn stover, 20% oat hay, 48.8% ground corn, 7% molasses, 1.2% urea, 1% soybean meal, 0.9% mineral premix,1.1% salt, dry matter basis) incubated with sheep ruminal fluid in 3 different occasions. Data were analyzed with PROC MIXED of SAS and orthogonal contrasts to determine the linear and quadratic effects of EM dose and exposure time. Interaction (P < 0.05) of EM x T was observed for in vitrodry matter digestibility (IVDMD), maximum gas volume (Vmax), total volatile fatty acids (VFA), acetate (ACE), propionate (PROP), butyrate (BUT) and ammonia-nitrogen (NH3), IVDMD was higher (P < 0.01, 4.8 and 3.72%) for T48EM1.5 than T12EM0 and T0EM0, PROP was higher (P < 0.05) for T48EM0, T48EM1 and 1.5 than T12EM0. The ACE:PROP ratio was higher (P < 0.05, 17.2%) for T12EM0 than T48EM1.5. IVDMD, PROP and NH3 linearly increased (P < 0.01) with increasing exposure time. EM levels have a quadratic effect (P < 0.01) with maximum response at EM0.5. It was concluded that the addition of 0.5 to 1.5 mL/kg DM of EM® to a sheep diet and increasing preincubation time, up to 48h, improve feed fermentation and digestibility.Project was supported by UNAM, DGAPA, PAPIIT (IT202120).