A psychrotrophic bacterium, Arthrobacter sp. ON14, isolated from Antarctica, was shown to exhibit a high β-galactosidase activity at a low temperature. A genomic library of ON14 was constructed and screened for β-galactosidase genes on functional plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as the substrate. Two different β-galactosidase genes, named as galA, galB, were found in ON14. Computational analyses of the genes revealed that the encoded protein GalA belongs to family 2 of glycosyl hydrolysases and is a cold-active protein, whereas GalB belongs to family 42 of glycosyl hydrolysases and is a mesophilic protein. Reverse transcription analyses revealed that the expression of galA is highly induced at a low temperature (4℃) and repressed at a high temperature (28℃) when lactose is used as the sole carbon source. Conversely, the expression of galB is inhibited at a low temperature and induced at a high temperature. The purified GalA showed its peak activity at 15℃ and pH 8. The mineral ions Na+, K+, Mg2+, and Mn2+ were identified as enzyme activators, whereas Ca2+ had no influence on the enzyme activity. An enzyme stability assay revealed that the activity of GalA is significantly decreased when it is incubated at 45℃ for 2 h, and all its activity is lost when it is incubated at 50℃.