The effects of the exogenous fibrolytic enzyme (ENZ) commercial preparation Dyadic® xylanase PLUS (Dyadic International, Inc., Jupiter, USA), containing endo-1,4-β-d-xylanase, on ruminal fermentation of maize stover, oat straw, and sugarcane bagasse were examined using the in vitro gas production (GP) technique. The ENZ commercial preparation was added at 0 (control), 60 (low), 120 (medium), and 240 (high) µg/g dry matter of substrate, and at two times of application (direct addition just before fermentation or with a 72-h pre-incubation before fermentation). Ruminal GP volumes were recorded at 2, 4, 6, 8, 10, 12, 14, 24, and 48 h of incubation, and substrate degradability and concentration of fermentation end-products (volatile fatty acids, ammonia, methane) in the cultures were determined at 48 h of incubation. Increased (P < 0.05) GP volume was observed in fibrous feeds treated with ENZ compared with untreated substrates. The pre-incubation vs the direct addition of ENZ resulted in greater GP volume (P < 0.05) with the three tested feeds. Enzyme addition decreased (P < 0.05) rumen pH compared to control when maize stover and sugarcane bagasse were incubated. Ammonia-N and total volatile fatty acids (VFA) were not affected (P > 0.05) by ENZ application in maize stover and oat straw. However, total and individual VFA concentrations, and CH 4 and CO 2 volumes were greater (P < 0.05) when sugarcane bagasse was incubated with 240 µg ENZ/g (P < 0.05). It can be concluded that the application of endo-1,4-β-d-xylanase enhances rumen fermentation of roughages, although the magnitude of the effects depends on the fibrous substrate fermented, the time of application, and the amount of enzyme added.