The development of viable gametes is especially susceptible to heat in all higher plants, including cotton (Gossypium hirsutum cv. Sicot 71), resulting in substantial reduction in lint quantity and quality at temperatures above 32 °C. Male reproductive cells are especially vulnerable to heat. This study demonstrates a relatively small impact of heat on leaves compared with a profound impact during early and late male gametophyte development. To investigate the mechanisms leading to heat sensitivity, the proteome of pollen was analyzed after two distinct phases of development (tetrads or binucleate microspores) had been exposed for 5 d to 36/25 °C (day/night) or 40/30 °C. The resulting mature pollen grains were collected for quantitative label-free shotgun proteomic analysis. A total of 868 proteins was quantified. Interestingly, Hsp70s were highly induced in response to extreme heat indicating key roles of this family to cope with heat stress.