This study examined the effect of shear velocity on biohydrogen producing dynamic membrane bioreactor (DMBR) containing 50 µm polyester mesh as supporting material. Increase of shear velocity up to 6.75 m/h enhanced hydrogen production performance as well as biomass retention in both suspended and attached forms, while wash-out was found at a shear velocity of 11.69 m/h. The highest average HPR, HY, suspended biomass, and attached biomass were 26.56 ± 1.49 L/L-d, 1.78 ± 0.10 mol H2/mol glucoseadded, 9.99 ± 0.11 g VSS/L, and 8.82 g VSS/L, respectively, at a shear velocity of 6.75 m/h. Flux balance analysis showed homoacetogenic pathway decreased at the shear velocity of 4.70 m/h with the increase of hydrogen yield based on consumed substrate. The highest copy numbers of Clostridium butyricum was found at the optimum shear velocity. Shear velocity would be a critical operational criteria for continuous biohydrogen production using DMBR.