Heterotrimeric guanine-nucleotide-binding proteins (G-proteins) consist of α, β and γ subunits and play important roles in response and tolerance to abiotic stresses in plants, but the function of the heterotrimeric G-protein β subunit in response to drought remains unclear. In the present study, the AGB1 mutants (agb1-2-1 and agb1-3-2) were more sensitive to drought than the wild-type. The overexpression of mulberry (Morus alba L.) G-protein β subunit in transgenic tobacco (Nicotiana tabacum L.) significantly enhanced the plants' drought tolerance. The transgenic tobacco plants had higher proline contents and peroxidase activities, and lower malonaldehyde and hydrogen peroxide contents and superoxide free radical accumulations under drought conditions. Additionally, transcript levels of the tobacco antioxidative genes, NtSOD and NtCAT, increased in drought-stressed transgenic tobacco plants. Thus, the heterotrimeric G-protein β subunits positively regulate drought tolerance in plants.