Anaerobic digestion of chicken manure produces biogas with an extreme H2S content (5000-6000 ppm), which greatly weakens the economic viability of biogas plants. In this study, in situ desulfurization through supplying limited air into the headspace of a digester and the impact upon the process performance were evaluated under different oxygen loads (1.4, 2.8, and 4.2 mL/gVS(in)) and injection approaches (single pulse and continuously). The results showed that an oxygen load at 4.2 mL/gVS(in) with continuous injection removed 99.7% of H2S (reduced to 10-15 ppm) and simultaneously increased the methane yield by 6.4%. Sulfide-oxidizing bacteria enrichment and the increased concentration of soluble iron were the main features of the air supplement system. The 16S rDNA results explained the attached growth of sulfur-oxidizing bacteria on the headspace carriers and the enrichment of Methanobacterium and Methanoculleus under the microaerated system. The obtained results suggest that in situ desulfurization technology could be applied more widely.