Our recent work showed the feasibility of using a 400 L pilot-scale bioreactor for hydrogen production via dark fermentation. However, the H 2 production performance of the pilot system was unsatisfactory when compared with that of lab-scale fermentors under the same conditions. This study applied engineering approaches to enhance the hydrogen production performance of the pilot bioreactor. First, a higher agitation rate was used to promote mass transfer efficiency. Next, the pilot system was operated under different combinations of hydraulic retention time (HRT) and substrate concentration (C S ) that gives different sets of organic loading rate (OLR) to improve bioH 2 production efficiency. With 25–30 rpm agitation rate and a OLR of 60 g COD/L/d (from combination of 8 h HRT and 20 g COD/L C S ), the H 2 production rate (HPR) of the pilot system reached 0.55 mol/L/d (13.4 m 3 /m 3 /d), which is 3.1 fold of that obtained from using a lower agitate rate (10–15 rpm). When operating at HRT = 6 h and C S = 30 g COD/L (i.e., OLR = 120 g COD/L/d), the pilot system obtained the highest HPR, hydrogen yield and overall hydrogen production efficiency of 1.18 mol/L/d, 3.84 mol H 2 /mol sucrose and 47.2%, respectively. This performance is similar to that obtained from the lab-scale system and is significantly higher than that from the original pilot tests prior to process optimization.