The effect of organic carbon source on the granulation of anaerobic ammonium oxidation (Anammox) sludge was tested in two parallel SBRs by gradually increasing the concentration of organic carbon source (R1) and by adding no organic carbon (R2). Results showed that appropriate concentration of organic carbon source (< 130 mg/L) increased the amount of extracellular polymer (EPS) in the reactor and consequently accelerated the formation of granular sludge and enhanced sludge settling performance. Sludge in the two groups was granulated at 28 and 35 days and the particle sizes were 450 μm and 409 μm, respectively. However, excessive organic carbon sources (> 230 mg/L) disintegrated sludge, reduced particle size, and worsened sludge settling performance significantly. Organic carbon source with a concentration lower than 110 mg/L promoted the anaerobic ammonia oxidation by denitrification, thereby improving N-removal efficiency. In contrast, when the concentration of organic carbon source was higher than 110 mg/L, anaerobic ammonia oxidation was inhibited and N-removal efficiency was decreased. Therefore, the concentration of organic carbon source should be equal to or lower than 110 mg/L during the operation of Anammox reactor.