In vitro models have been used to demonstrate that estrogen receptors (ERs) can regulate estrogen-responsive genes either by directly interacting with estrogen-responsive element (ERE) DNA motifs or by interacting with other transcription factors such as AP1. In this study, we evaluated estrogen (E₂)-dependent uterine gene profiles by microarray in the KIKO mouse, an in vivo knock-in mouse model that lacks the DNA-binding function of ER and is consequently restricted to non-ERE-mediated responses. The 2- or 24-h E₂-mediated uterine gene responses were distinct in wild-type (WT), KIKO, and ERKO genotypes, indicating that unique sets of genes are regulated by ERE and non-ERE pathways. After 2 h E₂ treatment, 38% of the WT transcripts were also regulated in the KIKO, demonstrating that the tethered mechanism does operate in this in vivo model. Surprisingly, 1438 E₂-regulated transcripts were unique in the KIKO mouse and were not seen in either WT or ERKO. Pathway analyses revealed that some canonical pathways, such as the Jak/Stat pathway, were affected in a similar manner by E₂ in WT and KIKO. In other cases, however, the WT and KIKO differed. One example is the Wnt/-catenin pathway; this pathway was impacted, but different members of the pathway were regulated by E₂ or were regulated in a different manner, consistent with differences in biological responses. In summary, this study provides a comprehensive analysis of uterine genes regulated by E₂ via ERE and non-ERE pathways.