We identified 102, 51 and 51 proteins encoded by the trihelix genes in Gossypium hirsutum, Gossypium arboretum and Gossypium raimondii, respectively. RNA sequence data and real-time quantitative polymerase chain reaction analysis showed thatGh_A05G2067(GT-2) was highly up regulated under drought and salt stress conditions. Transient expression ofGT-2-greenfluorescent protein fusion protein in protoplast showed thatGT-2was local-ized in the nucleus. The overexpression ofGT-2conferred an enhanced drought tolerance to cotton, with lower malondialdehyde, hydrogen perox-ide contents and higher reactive oxygen scavenging enzyme activities. More-over, chlorophyll content, relative leaf water content (RLWC), excised leaf water loss (ELWL) and cell membrane stability (CMS) were relatively sta-ble in theGT-2-overexpressed lines compared to wild-type (WT). Similarly, stress-responsive genes RD29A, SOS1,ABF4andCBL1were highly up regu-lated in theGT-2-overexpressed lines but were significantly downregulated in WT. In addition, theGT-2-silenced cotton plants exhibited a high level of oxi-dation injury, due to high levels of oxidant enzymes, in addition to negative effects on CMS, ELWL, RLWC and chlorophyll content. These results mark the foundation for future exploration of the trihelix genes in cotton, with an aim of developing more resilient, versatile and highly tolerant cotton genotypes.