A novel gene was isolated for the first time from a psychrophilic gram-negative bacterium Rahnella sp. R3. The gene encoded a cold-adapted β-galactosidase (R-β-Gal). Recombinant R-β-Gal was expressed in Escherichia coli BL21 (DE3), purified and characterized. R-β-gal belongs to the glycosyl hydrolase family 42. Circular dichroism spectrometry of the structural stability of R-β-Gal with respect to temperature indicated that the secondary structures of the enzyme were stable to 45°C. In solution, the enzyme was a homo-trimer and was active at temperatures as low as 4°C. The enzyme did not require the presence of metal ions to be active, but Mg2+, Mn2+, and Ca2+ enhanced its activity slightly, whereas Fe3+, Zn2+ and Al3+ appeared to inactive it. The purified enzyme displayed Km values of 6.5mM for ONPG and 2.2mM for lactose at 4°C. These values were lower than the corresponding Kms reported for other cold-adapted β-Gals.