Background: Cotton (Gossypium hirsutum) provides the largest natural fiber for the textile manufacturing industries, but its production is on the decline due to the effects of salinity. Soil salt-alkalization leads to damage in cotton growth and a decrease in yields. Hyperosmolality-gated calcium-permeable channels (OSCA) have been found to be involved in the detection of extracellular changes which trigger an increase in cytosolic free calcium concentration. Hyperosmolality-induced calcium ion increases have been widely speculated to be playing a role in osmosensing in plants. However, the molecular nature of the corresponding calcium ion channels remains unclearly. In this research work, we describe the OSCA genes and their putative function in osmosensing in plants by carrying out genomewide identification, characterization and functional analysis of the significantly up-regulated OSCA gene, GhOSCA1.1 through reverse genetics. Result: A total of 35, 21 and 22 OSCA genes were identified in G. hirsutum, G. arboreum, and G. raimondii genomes, respectively, and were classified into four different clades according to their gene structure and phylogenetic relationship. Gene and protein structure analysis indicated that 35 GhOSCA genes contained a conserved RSN1_7TM (PF02714) domain. Moreover, the cis-regulatory element analysis indicated that the OSCA genes were involved in response to abiotic stress. Furthermore, the knockdown of one of the highly up-regulated genes, Gh_OSCA1.1 showed that the virus-induced gene silenced (VIGS) plants were highly sensitive to dehydration and salinity stresses compared with the none VIGS plants as evident with higher concentration levels of oxidant enzymes compared with the antioxidant enzymes on the leaves of the stressed plants. Conclusion: This study provides the first systematic analysis of the OSCA gene family and will be important for understanding the putative functions of the proteins encoded by the OSCA genes in cotton. These results provide a new insight of defense responses in general and lay the foundation for further investigation of the molecular role played by the OSCA genes, thereby providing suitable approaches to improve crop performance under salinity and drought stress conditions.