We leveraged a human gut-on-a-chip (Gut Chip) microdevice that enables independent control of fluid flow and mechanical deformations to explore how physical cues and morphogen gradients influence intestinal morphogenesis. Both human intestinal Caco-2 and intestinal organoid-derived primary epithelial cells formed three-dimensional (3D) villi-like microarchitecture when exposed to apical and basal fluid flow; however, 3D morphogenesis did not occur and preformed villi-like structure involuted when basal flow was ceased. When cells were cultured in static Transwells, similar morphogenesis could be induced by removing or diluting the basal medium. Computational simulations and experimental studies revealed that the establishment of a transepithelial gradient of the Wnt antagonist Dickkopf-1 and flow-induced regulation of the Frizzled-9 receptor mediate the histogenesis. Computational simulations also predicted spatial growth patterns of 3D epithelial morphology observed experimentally in the Gut Chip. A microengineered Gut Chip may be useful for studies analyzing stem cell biology and tissue development.