SUMMARYWhen pollen lands on a receptive stigma, it germinates and extends a tube inside the transmitting tissue of the pistil to deliver the sperm cells for double fertilization. The growth of the pollen tube triggers significant alterations in its gene expression. The extent to which these changes occur in the vegetative cell or extend to the sperm cells transported by the tube is unclear but important to determine since sperm cells are believed to acquire a competency for fertilization during pollen–pistil interactions. To address these questions, we compared the transcriptomes of Arabidopsis thaliana sperm cells and vegetative nuclei isolated from mature pollen grains with those isolated from in vitro‐grown pollen tubes. Importantly, we also compared transcriptomes of sperm cells obtained from pollen tubes grown under semi‐in vivo conditions where tubes passed through a pistil section. Our data show that extensive transcriptomic changes occur in sperm cells during pollen tube growth, some of which are elicited only as sperms are carried through the pistil. Their analysis reveals a host of previously unidentified transcripts that may facilitate sperm maturation and gamete fusion. The vegetative cell undergoes even more extensive transcriptomic reprogramming during pollen tube growth, mainly through the upregulation of genes associated with pollen tube growth and vesicle‐mediated transport. Interestingly, ATAC‐seq data show that the promoters of genes upregulated in sperm during pollen tube growth are already accessible in sperm chromatin of mature pollen grains, suggesting pre‐configured promoter accessibility. This study's expression data can be further explored here: https://bar.utoronto.ca/eFP‐Seq_Browser/.