The study was conducted to evaluate the effects of microbial culture supplements on ruminal fermentation and fermentative quality of Italian ryegrass silage (IRGS) both in vitro and in situ. Three species of microbes (Lactobacillus casei (LC), Bacillus subtilis (BS), and Saccharomyces cerevisiae (SC)) were used in this study. They were applied to IRGS at 30 days after silage manufacture. Various items were measured using in vitro and in situ incubation technique after each microbial supplement was inoculated into IRGS at 0.5×104 CFU/g. In the first experiment, in vitro ruminal fermentation characteristics of IRGS were evaluated at 0, 12, 24, 48, and 72 hours after microbes were inoculated into IRGS. In the second experiment, in situ fermentation characteristics were investigated at 0, 1, 3, and 5 days after the inoculation of each microbial supplement. In vitro ruminal NH3-N content was significantly (p<0.05) increased in LC-, BS-, and SC-IRGS at 12 hrs post incubation compared to that in control IRGS. In vitro ruminal total VFA concentration and dry matter digestibility (DMD) of IRGS were not significantly difference among LC-, BS-, and SC-IRGS, although they were numerically increased in LC-IRGS than those of the other IRGS. In addition, this study evaluated the fermentation characteristics and in situ DMD of IRGS with the lapse of incubation time up to 5 days. Throughout the incubation times from 1 day to 5 days, the pH value was significantly (p<0.05) lower in BS-, LC-, and SC-IRGS than that in control IRGS. Lactate was significantly (p<0.05) higher, and significantly (p<0.05) butyrate was lower in LC-IRGS than that in other treatments at 0 day. It was higher (p<0.05) in control IRGS than that of BS-, LC-, and SC-IRGS at 1-5 days. In situ DMD tended to increase in BS-, LC-, and SC-IRGS compared to that in control IRGS. Especially, DMD was higher in SC-IRGS than that in other treatments at 0 day. It tended to be higher in LC-IRGS at all incubation time. Taken together, these results suggest that it might be useful to select a microorganism by considering the feeding time of IRGS to ruminants because organic acids and DMD of IRGS were affected by the incubation time of each microorganism with IRG silage, especially for L. casei decreased the content of acetate and butyrate in IRGS.