This study examined effects of enzyme type and duration of the enzyme-substrate interaction (preincubation) period before in vitro fermentation on the in vitro digestibility and gas and methane production of corn silage (CS). Exactly 1 mL of 0.1 M citrate phosphate buffer (pH 6.0; Control) or the buffer and either xylanase (XYL; 1 mg/g) or xylanase-cellulase (MIX 3.4 mg/g) fibrolytic enzyme preparations were incubated with 0.5 g of dried, ground CS at room temperature for 0, 12, 24 and 48 h in a 120-mL culture bottle. Subsequently, 40 mL of buffered-rumen fluid was added to each bottle and the contents were incubated for 24 h at 39°C. At least 3 replicates of each treatment combination were examined in each of 5 runs. Total gas and methane production, volatile fatty acids (VFA), pH and in vitro digestibility of dry matter (DMD) and neutral detergent fiber (NDFD) were measured. The data were analyzed as a completely randomized design with a 3 × 4 factorial treatment arrangement using the GLIMMIX procedure of SAS (SAS Inst. Cary, NC). There were no (P > 0.10) enzyme by time interactions for any measure except DMD (P = 0.09). Preincubating MIX for 12 or 24 h with CS resulted in less (P < 0.05) DMD than that at 0 h but similar DMD to that at 48 h. Preincubating XYL with CS for 12 h resulted in greater (P < 0.05) DMD than that at 0, 24 or 48 h. Both MIX and XYL increased NDFD but MIX had the greatest value (P < 0.05; 58.6 and 53.6 vs 51.0%, respectively). Compared with CON, MIX increased gas production (40.1 vs. 42.9 mL) and decreased methane production (P < 0.05; 0.023 vs. 0.019 mg/mL) but XYL had similar values to the other treatments (41.4 mL and 0.021 mg/mL, respectively). Across enzyme treatments, pH was greater after 12 h of preincubation (6.34 vs. 6.28) and gas production was greater after 48 h (45.5 vs. 40.17 mL) compared with other incubation times. The MIX enzyme was more effective than the XYL enzyme at increasing the fermentation and digestion of CS. Enzyme-substrate preincubation period had enzyme-specific effects on DMD and no effects on NDFD or other fermentation measures.