Ecological speciation is a common mechanism by which new species arise. Despite great efforts, the role of gene expression in ecological divergence and speciation is poorly understood. Here, we conducted a genome-wide gene expression investigation of two Oryza species that are evolutionarily young and distinct in ecology and morphology. Using digital gene expression technology and the paired-end RNA sequencing method, we obtained 21,415 expressed genes across three reproduction-related tissues. Of them, approximately 8% (1,717) differed significantly in expression levels between the two species and these differentially expressed genes are randomly distributed across the genome. Moreover, 62% (1,064) of the differentially expressed genes exhibited a signature of directional selection in at least one species. Importantly, the genes with differential expression between species evolved more rapidly at the 5′ flanking sequences than the genes without differential expression relative to coding sequences, suggesting that cis-regulatory changes are likely adaptive and play an important role in the ecological divergence of the two species. Finally, we showed evidence of significant differentiation between species in phenotype traits and observed that genes with differential expression were overrepresented with functional terms involving phenotypic and ecological differentiation between the two species, including reproduction- and stress-related characteristics. Our findings demonstrate that ecological speciation is associated with widespread and adaptive alterations in genome-wide gene expression and provide new insights into the importance of regulatory evolution in ecological speciation in plants.