A sequencing batch reactor was used to investigate the effect of carbon sources on the metabolism of activated sludge. Acetate and phenol, with the chemical oxygen demand (COD) of 330–350 mg L−1, was used as the carbon source in Periods I and II, respectively. Acetate decreased in the initial 120 min with the intracellular storage materials (XSTO), extracellular polymeric substances (EPS), and the soluble microbial products (SMP) accumulating to 131.0 mg L−1, 347.5 mg L−1, and 35.5 mg L−1, respectively. Then, XSTO and EPS decreased to 124.5 mg L−1 and 340.0 mg L−1, respectively, in the following 120 min. When acetate was replaced by phenol, it could not be used at the beginning due to its toxicity. The XSTO decreased from 142 mg L−1 to 54.6 mg L−1 during the aeration period. The EPS had a significant increase, with the highest value of 618.1 mg L−1, which then decreased to 245.6 mg L−1 at 240 min. The phenol was gradually degraded with the acclimation and it can be fully degraded 18 d later. Meanwhile, the usage ratio of the internal carbon source decreased. The effluent SMP in Period II was 1.7 times that in Period I.