The state and development of the intestinal epithelium is vital for infant health, and increased understanding in this area has been limited by an inability to directly assess epithelial cell biology in the healthy newborn intestine. To that end, we have developed a novel, noninvasive, molecular approach that utilizes next generation RNA sequencing on stool samples containing intact epithelial cells for the purpose of quantifying intestinal gene expression. We then applied this technique to compare host gene expression in healthy term and extremely preterm infants. Bioinformatic analyses demonstrate repeatable detection of human mRNA expression, and network analysis shows immune cell function and inflammation pathways to be up-regulated in preterm infants. This study provides incontrovertible evidence that whole-genome sequencing of stool-derived RNA can be used to examine the neonatal host epithelial transcriptome in infants, which opens up opportunities for sequential monitoring of gut gene expression in response to dietary or therapeutic interventions.