A cold shock domain (CSD)-containing protein, CspD, of molecular mass ∼7.28 kDa in a psychrotolerant Antarctic Janthinobacterium sp. Ant5-2 (ATCC BAA-2154) exhibited constitutive expression at 37, 22, 15, 4 and −1 °C. The cspD gene encoding the CspD protein of Ant5-2 was cloned, sequenced and analyzed. The deduced protein sequence was highly similar to the conserved domains of the cold shock proteins (Csps) from bacteria belonging to the class Betaproteobacteria. Its expression was both time- and growth phase-dependent and increased when exposed to 37 °C and UV radiation (UVC, dose: 1.8 and 2.8 mJ cm−2). The results from the electrophoretic mobility shift and subcellular localization study confirmed its single-stranded DNA-binding property. In silico analysis of the deduced tertiary structure of CspD from Ant5-2 showed a highly stable domain-swapped dimer, forming two similar monomeric Csp folds. This study established an overall framework of the structure, function and phylogenetic analysis of CspD from an Antarctic Janthinobacterium sp. Ant5-2, which may facilitate and stimulate the study of CSD fold proteins in the class Betaproteobacteria.