Serine hydroxymethyltransferase (SHMT) plays a significant role in the synthesis of L-Serine, purine and thymidylate, which could be extensively applied in the treatment of cancers and the development of antibiotics. In this study, cloned from Psychrobacter sp. ANT206, a novel cold-adapted SHMT gene (designated as psshmt, 1257 bp) encoding a protein of 418 amino acids was expressed in Escherichia coli. The homology modeling result revealed that PsSHMT owned fewer Pro residues and hydrogen bonds compared with its homologous mesophilic SHMT from Escherichia coli (EcSHMT). In addition, the molecular weight (MW) of the purified recombinant PsSHMT (rPsSHMT) was identified to be 45 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), approximately. The enzymatic characteristics of the cold-adapted rPsSHMT displayed that its optimum temperature and pH was 30 °C and 7.5, respectively, and its enzymatic activity could be inhibited by Cu2+, significantly. rPsSHMT also showed a high kcat value and low ΔG at low temperatures. Furthermore, Arg could affect the activity of rPsSHMT and be vital to its active sites. These results of this study reflected that these characteristics of the cold-adapted rPsSHMT made it a remarkable candidate that could be utilized in multiple industrial fields under low temperature.