The CBL/CIPK signaling system mediates a variety of responses to environmental stimuli in plants. In this work, we identified four CBL genes from Gossypium hirsutum, two of which (designated GhCBL2 and GhCBL3) showed preferential expression in the elongating fiber cells. Moreover, the expression patterns of these two CBL genes coincided with that of a putative CBL-interacting protein kinase gene (GhCIPK1) that we isolated in a previous study. Yeast two-hybrid assay indicated that among the four CBLs, GhCIPK1 interacted selectively with GhCBL2 and GhCBL3. The co-expression and interactions of these proteins suggest that they are components of the same signaling pathway. These findings strengthen our previous prediction that CBL/CIPK signaling plays a critical role in the regulation of cotton fiber elongation.