Two enhanced biological phosphorus removal (EBPR) reactors were started up at low temperatures to obtain microorganisms responsible for aerobic and anoxic phosphorus removal, namely polyphosphate-accumulating organisms (PAO) and denitrifying PAO (DPAO), and their operational performance and microbial community were together investigated in the hope of assessment of the effectiveness of the EBPR process at low temperature by combining chemical analysis and microbial community structure evolution based on polymerase chain reaction-denaturing gradient gel electrophoresis. When two reactors reached the steady state after 40 and 80 days for the anaerobic–aerobic (AO) and anaerobic–anoxic (AA) reactor operation in AO and AA modes, respectively, a good ability of anaerobic phosphorus release and aerobic or anoxic phosphorus uptake was present both in these two reactors. During this start-up process, a total of 22 bands were detected in seed, AA and AO sludge samples, including Alpha-, Beta-, Gamma- and Deltaproteobacteria, as well as Chlorobi, Firmicutes, Bacteroidetes and Actinobacteria. Of all the bands, only four bands were present in all the lanes, suggesting that shift in microbial community occurred greatly depending on the electron acceptors in this study. From evolutionary tree, it was found that microorganisms related to DPAO mostly belong to the phylum Betaproteobacteria, while microbes corresponding to PAO were present in several phyla. Overall, the new strategy proposed here was shown to be feasible for the enrichment of PAO and DPAO at low temperature, and may be regarded as a new guidance for the application of EBPR technology to practice, especially in winter.