This study evaluated if hydrolysis by exogenous fibrolytic enzymes (EFE) can be synergistically improved by recombinant bacterial expansin (BsEXLX1) and examined if the expansin alone can expand plant cell walls. In Experiment 1 (E-1), the objective was to examine if EFE (2.3 mg/g) and BsEXLX1 (162 µg/g) synergistically hydrolyze filter paper (FP; 6 mg) and xylan (20 mg). The substrates were incubated in quadruplicate in 3 runs for 12h at pH 6 and 39°C and reducing sugars were assayed using Control, EFE, BsEXLX1 and EFE + BsEXLX1. In Experiment 2 (E-2), the objective was to verify disruptive activity of BsEXLX1 (162 µg/ g) and control (Buffer only) on cotton fibers (20 mg) incubated in triplicate for 1 h at pH 4 and 50°C. Cell wall extension was measured and immunofluorescence was used to localize BsEXLX1. Experiment 3 (E-3) examined the existence of synergistic effects of EFE (2.3 mg/ g) and BsEXLX1 (162 µg/ g) on in vitro ruminal degradability and preingestive hydrolysis of bermudagrass silage (0.5 g). Dried ground (1 mm) silage samples were incubated with nothing, BsEXLX1, EFE or EFE + BsEXLX1 in quadruplicate for 24 h in each of 3 runs. Data were analyzed using the NMLE package of R and a model that included fixed effects of EFE, BsEXLX1, run and interactions (E1) or random effect of cow nested within run (E-3). In E-1, the amount of sugars released from FP was 12% greater (P < 0.01) with EFE+BsEXLX1 compared with EFE (25.9 vs 23.2 mg/g FP). However, adding BsEXLX1 did not improve (P = 0.7) xylan hydrolysis by EFE (130.6 vs 129.2 mg/g xylan). In E-2, on average, BsEXLX1 expanded the diameter of cotton fiber cell walls by 30% (P < 0.01) compared with the Control and immunofluorescence confirmed the expansion was due to BsEXLX1. In E-3, EFE improved DM degradability in buffered rumen fluid by 4% (47.5 vs 45.7%, P < 0.01) and preingestive hydrolysis by 11% (20.6 vs 18.5%, P < 0.01); however synergism with BsEXLX1 was only detected for the latter (21.2 vs 20.1%, P = 0.02). Therefore, BsEXLX1 can expand plant cell walls and synergistically improve the hydrolysis of EFE in a substrate-dependent manner.