As with all flowering plants, the formation of gametes in cotton (Gossypium hirsutum) is highly susceptible to heat. Substantial reduction in lint quantity and quality occur when developing flowers and bolls are exposed to sustained temperatures above 32 oC, particularly as a result of the high vulnerability of plant male reproductive cells to heat stress. This study reports proteomic changes in the pollen of cotton (cultivar Sicot 71), aiming to identify heat-responsive proteins in the early and late male gametophyte. Two distinct phases of pollen development (tetrads and binucleate microspores) were exposed to 36/25 oC (day/night) or 40/30 °C, each for 5 days. Subsequently, mature pollen grains were collected for quantitative label-free shotgun proteomic analysis. A total of 868 proteins was identified across all samples analysed. Differential expression analysis demonstrated that 48 proteins were up-regulated in mature pollen after the exposure of squares at the tetrad stage to 36/25 °C, while 56 proteins were down-regulated. Severe cell damage was observed when squares were exposed to 40/30 °C at the tetrad stage, resulting in failed dehiscence. Moderate heat (36/25 oC) at the later binucleate microspore stage resulted in up-regulation and down-regulation of 47 and 24 proteins in the mature pollen grains, respectively, while extreme heat (40/30 °C) led to only ~30 proteins being differentially expressed. These differentially expressed proteins (DEPs) have functions related to metabolic process, catalytic activity, cellular process, binding, biological regulation, regulation of biological process, in addition to response to stimulus. The data revealed that heat shock proteins (HSPs) increased even long after heat stress. Interestingly, HSP70, one of the important HSPs, was up-regulated more than 100-fold under extreme heat stress.