Plants with highly reduced male gametophytes represent a successful adaptation to sexual reproduction, which plays an important role in the colonization and radiation of terrestrial ecosystems. During pollen maturation, microsporocytes to mature pollen grain cells switch from mitosis to meiosis and ultimately form a haploid male gametophyte, a widely used model to study plant development. We performed RNASeq analysis of Nicotiana tabacum at six developmental stages from microspores to mature pollen grain to characterize in detail key transcription factor (TF) genes involved in pollen ontogeny. Our results provide the most complete transcriptomic data during pollen development in the important model plant Nicotiana tabacum. We have identified DEGs associated with six ontogenetic stages of the male gametophyte, providing insights into the molecular regulation of reproductive development by TFs that have a high potential for agronomic research investigating molecular networks associated with pollen sterility and related issues.