Iron as micronutrients is of great significance for forming a stable and efficient anaerobic digestion of food waste. Aim of this study was to examine the effect of iron supplementation on the mesophilic anaerobic digestion of food waste. Firstly, batch experiments were conducted with different iron concentration at a constant kitchen waste/inoculum ratio (K/I = 1.0), which indicated that the effect of iron on anaerobic digestion of was strictly dosage-dependent. Then, anaerobic digestion of food waste was conducted for 50 days in semi-continuous rectors with optimal iron concentration (2.0mg /L) under the same conditions. And the semi-continuous rectors obtained a good operation performance with low volatile fatty acids concentration, higher biogas production, high coenzyme F420 and dehydrogenase concentrations. Furthermore, two samples taken on 7th day and 50th day were analyzed by high-throughput sequencing, which illustrated that the composition anaerobe community was stable. However, the growth and activity of several syntrophic microbial groups (Aminobacterium, Syntrophomonas, Anaerolineaceae, Methanosaeta, Methanosarcina, Methanobacterium and Methanospirillum), were stimulated by iron supplementation. The shift of microbial community suggested that a high-efficiency microbial community for methane production from food waste was formed by iron supplementation.