The intestinal epithelium serves as a major site of dietary nutrient absorption and microbial interactions. These important roles for intestinal epithelial cells (IECs) are shared among all vertebrates, but underlying transcriptional regulatory mechanisms are unresolved. Here we use a comparative functional genomics approach to test the hypothesis that conserved IEC functions are achieved using conserved transcriptional regulatory mechanisms across vertebrate species. We generated genome-wide adult IEC RNA-seq and open chromatin data (FAIRE-seq or DNase-seq) from four vertebrate species: Zebrafish, Stickleback, Mouse, and Human. Despite the evolutionary distance between these species, we observe moderate correlations between relative IEC mRNA levels of 1to1to1to1 orthologs and identify a core subset of orthologous genes comprising a vertebrate IEC signature. We also compare existing expression data of mouse and zebrafish and find additional evidence for transcriptional and functional conservation regionally along the length of the intestine. Despite conservation of expression patterns, we find that only a few open chromatin regions overlapping conserved noncoding elements (CNEs) are specifically accessible in IECs. Using transgenic reporter assays to test the regulatory potential of these regions, we identified uncharacterized CNEs neighboring orthologous hes1/her6 loci that are sufficient to drive IEC expression in zebrafish. However, these highly conserved IEC-specific open chromatin regions are very rare, and do not explain the tissue-specific expression of most IEC genes. In contrast, we find that many non-conserved regions neighboring orthologous genes in different genomes can still drive highly similar IEC expression in zebrafish. This indicates that traditional sequence conservation metrics do not sufficiently capture cis-regulatory information that is similar between species. In accord, we identify a set of transcription factor binding motifs that are similarly enriched in open regulatory regions near IEC signature genes despite a lack of traditional sequence conservation, suggesting a conserved transcriptional regulatory network. This work establishes a genomic resource for comparing and dissecting these transcriptional regulation networks that underlie shared intestinal structure and function in vertebrate species.