The plant U-box (PUB) proteins function as E3 ligases to poly-ubiquitinate the target proteins for their degradation or post-translational modification. PUBs also play important roles in regulation of diverse biological processes, including plant response to environmental stresses. In this study, the functional characterization of a soybean PUB gene, GmPUB6, was performed. GmPUB6 was mainly localized to peroxisome, and showed E3 ubiquitin ligase activity. The transcript levels of GmPUB6 in soybean leaves and roots were induced by abscisic acid (ABA), high salinity and polyethylene glycol (PEG) treatment. Comparing with the wild-type (WT) plants, overexpression of GmPUB6 in Arabidopsis thaliana decreased plant survival rate after drought stress, reduced seed germination rate and root elongation under mannitol (osmotic) stress, and suppressed ABA- or mannitol-mediated stomatal closure. In addition, under dehydration stress, the relative expression levels of seven stress responsive genes, including ABI1, DREB2A, KIN2, RAB18, RD20, RD29A and RD29B, were lower in GmPUB6-overexpressed plants than WT. Taken together, these results suggest that GmPUB6 functions as a negative regulator in drought tolerance, and plays an important role in osmotic stress and ABA signaling pathways, which might be the possible mechanism of PUB6 participating in drought stress response.