Male sterility is used in the production of hybrid seeds of several crops and can effectively improve the breeding efficiency of cotton hybrids. Reactive oxygen species (ROS) is closely associated with the tapetum and pollen development, but their relationship in cotton male fertility remains unclear. In this study, we comprehensively compared the cytology and proteomics of the anthers from an Upland cotton (Gossypium hirsutum) material, Shida 98 (WT), and its nearly-isogenic male sterile line Shida 98A (MS). Cytological observations indicated delayed PCD in the tapetum and defects in microspore development in MS anthers. Further results revealed that the ROS homeostasis in MS anthers was disrupted. Proteomic results showed that proteins differentially expressed between WT and MS anthers were mainly related to redox homeostasis, protein folding, and apoptotic signaling pathways. HSP70 and APX1 were identified as the two key proteins during cotton anther development. Exogenous application of HSP70 inhibitor increased the level of H2O2 and decreased the activity of APX1 and pollen viability in anthers. Altogether, the proteomics results imply HSP70 and APX1 being the key players jointly regulating tapetal PCD and pollen development by mediating ROS homeostasis. These results provide insights into the proteins associated with male fertility in cotton.