Brassinosteroids (BRs) are plant steroid hormones that not only play vital roles in plant growth and development, but also in mediating stress responses. A group of calmodulin-binding proteins, known as CBP60s are also involved in mediating the response of plants to stress. The aims of the present study were: (1) to investigate the effect of exogenous 24-epibrassinolide (EBR) on the phenotype of cotton (Gossypium hirsutum) seedlings under mild to moderate biotic and abiotic stresses, (2) to find and characterise cotton CBP60-encoding genes, orthologous to Arabidopsis CBP60s with known involvement in stress responses, and to investigate whether EBR may act by modulating the expression of GhCBP60 genes in cotton leaf tissue under salt stress. Experiments were designed to demonstrate the effects of EBR application from 0.1 to 2 µM on the phenotypic responses of cotton seedlings to mild/moderate salt, drought and pathogen (Verticillium dahliae) stresses. Results show that the exogenous application of EBR at low concentrations of 0.1 and 0.2 µM had no positive effect on seedling growth under all stresses. In addition, EBR at a higher concentration (0.5 µM) or with the surfactant Tween 20 caused toxic effects. Bioinformatics approaches revealed the presence of GhCBP60 orthologues of AtCBP60. Phylogenetic analysis indicated that CBP60a, CBP60g, andSARD1 from Arabidopsis each have four co-orthologues in cotton. AtCBP60f has two coorthologues, whereas CBP60b/c/d have nine co-orthologues. Multiple amino acid sequence alignments indicate that the DNA-binding and CaM-binding domains of AtCBP60 are highly conserved in GhCBP60, suggesting similar protein structures to AtCBP60. Prediction of subcellular localisation suggested that all GhCBP60 proteins contain a nuclear localisation signal. This, together with the highly conserved putative DNA binding region, suggests that all GhCBP60 are transcription factors. The results of qRT-PCR demonstrated that EBR treatment of cotton up-regulated the expression of GhCBP60a/f/g. On the other hand, salt down-regulated the expression of GhCBP60a but up-regulated the expression of GhCBP60f/g. Interestingly, treatment with EBR in the absence of salt restored the expression of GhCBP60a to levels similar to the control tissue. Analysis of promoters of GhCBP60 genes for putative BR-related transcription factor binding motifs indicated the presence of CANNTG and GGTCC elements. However, these were not significantly enriched in stress-regulated genes. Furthermore, higher stringency BR-signalling-related elements: BRRE (CGTGTG/CGTGCG), G-box (CACGTG) and transcription factors TGA 1/TGA4 (TGACG) sense strands were absent in stress responsive genes GhCBP60a/f/g and GhSARD1 as compared to other groups. In the light of these results, I concluded that brassinosteroids (BRs) positively regulates the expression of novel GhCBP60 genes suggesting a possible connection between BR signalling and GhCBP60 transcription factors in mediating abiotic stress responses in cotton. However, the results from the cis-element search suggest that this connection is likely to be indirect rather than via a direct interaction with the BR signal transduction pathway.