Discussions on the positive effect of nano-sized metal addition to dark fermentation biohydrogen production have been raised but the real stimulation mechanism remains unclear. In this study, biohydrogen production enhancement by nanoparticle metal addition was tested using a strain of a known hydrogen producer, Clostridium pasteurianum. Gene expression and growth activity were evaluated. Biochemical hydrogen potential tests on the added TiO2 and Fe nanoparticles were performed at 35 °C with various metal concentrations (control and 50, 100, 200, 400, and 800 ppm). Comparison with the control showed that adding 50 ppm Fe nanoparticles could significantly increase the hydrogen production, which was expressed H2 gas volume, by 24.9%. The corresponding hydrogen production rate increased to 8.7 H2-L/L-d. This positive stimulation effect gradually decreased with increasing metal concentrations added. The effect eventually caused inhibition when the metal concentration reached 400 and 800 ppm. The highest maximum hydrogen production rate (Rmax) and the potential hydrogen production (P) in the simulation kinetic model were 45.2 mL/h and 255.7 mL, respectively. Despite increase in the gas production, metal addition did not increase the overall hydrogen yield (mol H2/mol xylose). Hence, this stimulation may not occur on the microorganism metabolism level. Analysis of gene expression indicated that addition of Fe nanoparticles did not remarkably improve the hydrogen enzyme activity of C. pasteurianum. Overall, hydrogen production stimulated by adding nano-metals was not directly related to enzyme activity improvement. •Addition of magnetic hematite nanoparticle could increase hydrogen production of dark fermentation.•Addition of NP-Fe did not remarkably improve enzyme activity despite an improvement of hydrogen gas production.•Hydrogen production stimulated by adding nano-metals might due to the enhancement of electron transfer.