A metabolic uncoupler, tetrakis (hydroxymethyl) phosphonium sulfate (THPS), was used to reduce sludge production in a laboratory-scale anaerobic/anoxic/oxic (A2/O) process. THPS was continuously added into anaerobic, anoxic, and oxic tank of the systems, respectively, along with 3.5 mg/L influent. The effects of THPS additions on sludge reduction, wastewater treatment efficiencies, and sludge characteristics were discussed in about a 99 d operational period. The results showed that the cumulative reduction of waste activated sludge reached about 30.8–31.7% and the decrease of sludge yield (Yobs) reached about 16.1–16.5% with the system added THPS in the anaerobic tank the most. Meanwhile, the wastewater treatment efficiency decreased slightly when THPS was added. Analysis of microbial communities in activated sludge in the oxic tank suggested that adding THPS would result in a new dominant micro-organism. However, the efficiencies of sludge reduction and wastewater treatment and microbial communities of activated sludge were similar when THPS was added into different tanks of the A2/O process, so the adding site of the metabolic uncoupler had little effect.