Cucumber (Cucumis sativus L.) predominately translocates raffinose family oligosaccharides (RFOs) in the phloem and also accumulates RFOs in leaves under stress. Galactinol synthase (GolS) catalyzes the critical step of RFOs biosynthesis and it is interesting to know the expression pattern and function diversity of multiple GolS isoforms in cucumber. In this study, we found all four CsGolS transcripts were up-regulated by different abiotic stresses. β-glucuronidase staining and tissue separation experiments suggested that CsGolS1 expresses in vascular tissues while other three CsGolSs are located in mesophyll cells. Phylogenetic analysis revealed that except Cucurbita, GolS promoters of all other cucurbits in group I own intermediary cell (IC)-specific cis-sequences, indicating this group is responsible for RFOs loading. Further investigation indicated that CsGolS1 plays double roles in both assimilate loading and stress adaptation in the IC, which could increase the RFOs concentration in the phloem sap and then improve the assimilate transport under adverse conditions. Cold-induced IC-specific overexpression of CsGolS1 enhanced the assimilate translocation efficiency and accelerated growth rates of sink leaves, fruits and whole plants under cold stress. Finally, our results demonstrate an unique mechanism of cucumber to adapt adverse environment and provide a potential biotechnological strategy to improve stress resistance of cucurbits.