Substrate utilization in a glucose-fed anaerobic sequencing batch reactor (ASBR) under different F/M ratios was investigated. Glucose added was quickly taken up by fermentation bacterial in anaerobic digestion, then partly degraded directly to volatile fatty acid (VFA, accounted for 34%-38% of COD-fed) and partly accumulated transiently in the cell as glycogen (accounted for 41%-46% of COD-fed) and degraded to VFA in the following. The bacterial accumulation capacities increased with F/M. The maximum specific storage capacities could reach as high as 116.8, 81.1 and 62.4 mg/g as F/M (COD/VSS) was controlled in 0.27, 0.20 and 0.14 respectively. VFA production rate slowed down due to glucose storage, which resulted in low VFA accumulation and guaranteed a stable operation in ASBR under high loading rate.