Corn stover has great potential utility as a ruminant feed because it contains large proportions of cellulose and hemicellulose. The purpose of this study was to characterize the saccharification, fermentation, and microbial colonization of steam-exploded corn stover. The steam explosion parameters for corn stover were optimized using a Box–Behnken design, based on 3 factors: steam pressure (1.0, 1.6, or 2.2 MPa), processing time (30, 115, or 200 s), and moisture content (10%, 30%, or 50%). The chemical components and morphological structure of steam-exploded corn stover were analyzed. The in sacco incubation of steam-exploded corn stover was assessed with nylon bags in the rumen of 4 dairy cows, in which steam-exploded corn stover was incubated for 48 h, analyzed for degradation, and examined with scanning electron microscopy (SEM) to observe the microbes present. The software Design-Expert was used for optimum levels statistics. Date were analyzed using the one-way ANOVA procedure of SAS. The optimal steam explosion conditions were 1.51 MPa, 180 s, and 10% moisture, insofar as they maximized the gas production rate during rumen fermentation in vitro. Steam explosion significantly reduced the content of neutral detergent fiber (NDF) and hemicellulose in the corn stover by 14.9% and 54.3%, respectively, and increased the production of total reducing sugars and xylose. SEM showed that the steam-exploded corn stover became more disordered and looser, suggesting that it is more accessible to rumen microbes. Fourier transform infrared spectroscopy indicated that the removal of hemicellulose and the abundance of cellulose increased after steam explosion. After rumen incubation for 4, 24, 36, or 48 h, the steam-exploded corn stover had higher cellulose and hemicellulose degradation (P < 0.05) than the raw corn stover. SEM showed that steam explosion markedly increased the bacterial colonization and biofilm formation on the surface of the corn stover. In conclusion, steam explosion facilitates the attachment of microbes to corn stover and its degradation in the rumens of dairy cows.