A 10 L upflow microbial fuel cell (UMFC) was constructed for simultaneous carbon and nitrogen removal. During the 6-month operation, the UMFC constantly removed carbon and nitrogen, and then generated electricity with synthetic wastewater as substrate. At 5.0 mg L−1 dissolved oxygen, 100 Ω external resistance, and pH 6.5, the maximum power density (Pmax) and nitrification rate for the UMFC was 19.5 mW m−2 and 17.9 mg·(L d)−1, respectively. In addition, Pmax in the UMFC with chicken manure wastewater as substrate was 16 mW m−2, and a high chemical oxygen demand (COD) removal efficiency of 94.1% in the UMFC was achieved at 50 mM phosphate-buffered saline. Almost all ammonia in the cathode effluent was effectively degraded after biological denitrification in the UMFC cathode. The results can help to further develop pilot-scale microbial fuel cells for simultaneous carbon and nitrogen removal.