Separate sexes and sex-biased gene expression have repeatedly evolved in animals and plants, but the underlying changes in gene expression remain unknown. Here, we studied a pair of plant species, one in which separate sexes and sex chromosomes evolved recently and one which maintained hermaphrodite flowers resembling the ancestral state, to reconstruct expression changes associated with the evolution of dioecy. We found that sex-biased gene expression has evolved in autosomal and sex-linked genes in the dioecious species. Most expression changes relative to hermaphrodite flowers occurred in females rather than males, with higher and lower expression in females leading to female-biased and male-biased expression, respectively. Expression changes were more common in genes located on the sex chromosomes than the autosomes and led to feminization of the X chromosome and masculinization of the Y chromosome. Our results support a scenario in which sex-biased gene expression evolved during the evolution of dioecy to resolve intralocus sexual conflicts over the allocation of resources.