A green synthesis of iron oxide nanoparticles (FeNPs) was developed using Murraya koenigii leaf extract as reducing and stabilizing agent. UV-vis spectra show that the absorption band centred at a wavelength of 277 nm which corresponds to the surface plasmon resonances of synthesized FeNPs. Fourier transform infrared spectroscopy spectrum exhibits that the characteristic band at 580 cm(-1) is assigned to Fe-O of gamma-Fe2O3. Transmission electron microscopy image confirms that the spherical with irregular shaped aggregates and average size of nanoparticles was found to be similar to 59 nm. The effect of synthesized FeNPs on fermentative hydrogen production was evaluated from glucose by Clostridium acetobutylicum NCIM 2337. The hydrogen yield in control experiment was obtained as 1.74 +/- 0.08 mol H-2/mol glucose whereas the highest hydrogen yield in FeNPs supplemented experiment was achieved as 2.33 +/- 0.09 mol H-2/mol glucose at 175 mg/L of FeNPs. In addition, the hydrogen content and hydrogen production rate were also increased from 34 +/- 0.8 to 52 +/- 0.8 % and 23 to 25.3 mL/h, respectively. The effect of FeNPs was compared with supplementation of FeSO4 on fermentative process. The supplementation of FeNPs enhanced the hydrogen production in comparison with control and FeSO4. The supplementation of FeNPs led to the change of the metabolic pathway towards high hydrogen production due to the enhancement of ferredoxin activity. The fermentation type was shifted from butyrate to acetate/butyrate fermentation type at the addition of FeNPs.