Thiosulfinate, a nature antibiotic, existed in all parts of Allium thereby accumulating in kitchen waste vastly. However, few literatures were available related to its influence on volatile fatty acids (VFA) and hydrogen production when kitchen waste digestion technology was applied. This study aimed to explore the inhibitory effect and the relevant mechanism. Experimental results showed that the hydrogen accumulation decreased from 23.2 ± 0.8 to 8.2 ± 0.1 mL/g VSS (volatile suspended solid) and maximal total VFA yield decreased from 765.7 ± 21.2 to 376.4 ± 21.7 mg COD (chemical oxygen demand)/g VSS when the dosage of thiosulfinate increased from 0 to 12.5 µg/g VSS. The mechanism study indicated, compared with control group, that the butyric acid decreased from 59% to 20.1% of total VFA yield when reactor in present of 12.5 µg/g VSS thiosulfinate. Moreover, the relative activities of functional enzymes were inhibited 73.4% (butyryl-CoA) and 72.7% (NADH), respectively.