Cysteine-rich transmembrane module (CYSTM) peptides, which are widely distributed and highly conserved in eukaryotes, are largely involved in stress response and defence. However, the role of cotton CYSTM genes in the stress response has not been functionally characterized. In this study, we identified GhCYSTM9 as a cold stress-responsive CYSTM member from upland cotton. Compared with that in control cotton plants, GhCYSTM9 silencing in cotton resulted in reduced tolerance under cold stress, accompanied by higher MDA contents and lower proline contents and SOD activities in leaves. Overexpressing GhCYTMS9 in Arabidopsis significantly increased the seed germination rates and root elongation at the germination stage. Compared with wild-type seedlings, GhCYSTM9-overexpressing seedlings presented lower MDA contents and greater proline contents in leaves under cold stress. Transcriptome analysis of transgenic Arabidopsis revealed that GhCYSTM9 may contribute to the cold response by regulating oxidative stress-related genes to mediate ROS levels. Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed that GhCYSTM9 interacted with the light-harvesting chlorophyll a/b-binding protein GhLHBC2A1. Overall, our results revealed a positive role of GhCYSTM9 in cold stress defence and suggested candidate genes for the genetic breeding of cold defence.