For successful double fertilization in flowering plants (angiosperms), pollen tubes deliver two non-motile sperm cells towards female gametes (egg and central cell, respectively). Heatwaves, especially during the reproduction period, threaten male gametophyte (pollen) development, resulting in severe yield losses. Using maize (Zea mays) as a crop and grass model system, we found strong seed set reduction when moderate heat stress was applied for two days during the tetrad, uni- or bicellular stages of pollen development. We show that heat stress accelerates pollen development, reduced starch content, decreased enzymatic activity, and impairs pollen germination capabilities when applied at the tetrad or unicellular stage resulting in sterility. Heat stress at the bicellular stage impairs sperm cell development and transport into pollen tubes. Using maize marker lines, we analyzed the transcriptomes of isolated sperm cells heat stressed at the bicellular stage. Heat stress affected the expression of genes associated with transcription, RNA processing and translation, DNA replication, and the cell cycle. This included the genes encoding centromeric histone 3 (CENH3) and α-tubulin. Most genes that were mis-regulated encode proteins involved in the transition from metaphase to anaphase during pollen mitosis II (PM II). Heat stress also activated spindle assembly check point and meta- to anaphase transition genes in sperm cells. In summary, mis-regulation of the cell cycle and its developmental regulators during heat stress at the bicellular stage results in inhibition of sperm cell development and transport defects ultimately leading to sterility.