Trehalose-6-phosphate synthase (TPS) plays an important role in metabolic regulation and stress responses in a variety of organisms. However information about cotton TPS is poor. Here a cotton TPS gene GhTPS11 was isolated and characterized. Expression analysis revealed that GhTPS11 was induced in 20-day old cotton seedlings by heat drought and high salt stresses as well as GA and ABA. Moreover GhTPS11 was induced by chilling stress and mannitol while was depressed by sucrose. Tissue expression analysis indicated that GhTPS11 expressed higher in leaves than in stems and roots of 20-day old cotton seedlings. The GhTPS11 overexpressing Arabidopsis seeds germinated slower than the wild-type (WT) under chilling stress. Trehalose-6-phosphate (T6P) and trehalose contents were evidently higher in GhTPS11 overexpressing lines 3, 5, and 22 than in WT under normal germination condition as well as chilling stress. Further analysis demonstrated that the expression of ICE1 CBF3 and RCI2A was induced lower whereas that of CBF1 and CBF2 was induced higher under chilling stress in the GhTPS11 overexpressing seeds than WT respectively. These results suggested that GhTPS11 encoded a stress-responsive TPS protein and functioned in chilling stress during seed germination. Perhaps the chilling stress sensitivity of transgenic Arabidopsis seeds was caused by the expression changes of at least some chilling-related genes such as ICE1 CBFs and RCI2A other than HOS1. So this article provided the useful information for GhTPS11 usage for crop molecular breeding.