Plant G protein γ subunits have multiple functions in growth and development processes and in abiotic stress responses. Few functions of Gγ in horticultural crops have been revealed thus far. In this study, the potential function of CsGG3.1-2, one of the two alternative splice variants of Gγ gene CsGG3.1 in cucumber (Cucumis sativus L.), was investigated using transgenic plants overexpressing antisense CsGG3.1-2 under the control of the 35S promoter. The tolerance to chilling stress in transgenic plants was significantly decreased. Cold stress-related physiological parameters and the expression of CBFs and their downstream target genes were then measured. Compared with WT, the maximum efficiency of photosystem II (Fv/Fm), antioxidative enzymes activities, soluble protein, and proline accumulation decreased significantly in transgenic plants treated with cold stress, whereas the malonaldehyde (MDA) content increased. However, the overexpression of antisense CsGG3.1-2 did not affect the induction of cold-inducible genes. Quantitative real-time PCR (qPCR) analysis showed the increased expression of CBF genes and their downstream target genes in transgenic plants, suggesting that CsGG3.1-2 affects cold responses via CBF-independent pathways in cucumber. At the same time, the sucrose and fructose contents decreased in transgenic plants under both normal and cold conditions. These findings suggest that soluble sugar deficiency is associated with chilling sensitivity in transgenic plants, and CsGG3.1-2 may have a role in regulating carbohydrate metabolism in cucumber.