Hydrogen production from the anaerobic fermentation of crude glycerin is studied in order to value-add the surplus glycerin by-product from biodiesel process. Crude glycerin (CG) from biodiesel production is partially cleaned up and fed to the anaerobic fermentations by Enterobacter aerogenes and a mixed culture of microorganisms (from an industry biogas production unit). The contaminant removed from CG is a mixture of matter organic non-glycerin (MONG) that can reduce the efficiency of converting crude glycerin to value-added products. Removing MONG from CG can be simply and inexpensively done by the acidification of CG resulting in a MONG recovery of 75% and CGMR (Crude glycerin with MONG removed) with a MONG content of 6.43%. From the anaerobic fermentations, the effect of substrate inhibition is found. Ethanol is the main product from the fermentation by E. aerogenes. When refined glycerin and CGMR are used with the initial glycerin concentration of less than 30 g/l, the anaerobic digestions by E. aerogenes produce comparable results of glycerin conversion and ethanol yield. However, glycerin initial concentration of 10g/l is required to achieve a glycerin conversion of higher 90%. The fermentation of CG does not give competitive experimental results. Meanwhile, the fermentation of CGMR by a mixed culture of microorganisms is studied with an initial glycerin concentration of about 12 g/l in tap water and no additional supplement nutrition is applied. As a result, the hydrogen yield can be increased by 7.25 times and hydrogen content in the gas product is enhanced to about 83% when sludge is heat-pretreated before use. The maximum hydrogen yield obtained is 0.0029 g H2/g glycerin when the heat-pretreatment can enrich the hydrogen-producing bacteria. In conclusion, the fermentation of CGMR introduces the possibility of using CGMR in the biological process of converting glycerin to value-added products. Furthermore, if the recovered MONG is a regained biodiesel feedstock, use of CGMR will help increase the efficiency of natural resource usage.