Brush borders of intestinal epithelial cells are mandatory for nutrient uptake. Yet, which actin nucleators are crucial for forming the F-actin bundles supporting microvilli and the actin filaments of the terminal web, in which microvilli are rooted, is unknown. We show that mice lacking the actin nucleator Cobl surprisingly did not display reduced microvilli densities or changes in microvillar F-actin bundles or microvilli diameter but particularly in the duodenum displayed increased microvillar length. Interestingly, Cobl-deficient mice furthermore showed a significant widening of the terminal web. Quantitative analyses of high-resolution cryo-scanning electron microscopy (EM) of deep-etched duodenum samples revealed that Cobl is specifically important for the formation of fine filaments in the central terminal web that connect the apical structure of the terminal web underlying the plasma membrane, the microvilli rootlets and the basal structure of the terminal web with each other. Thus, the actin nucleator Cobl is critically involved in generating one of the cellular structures of the brush border-decorated apical cortex of enterocytes representing the absorptive intestinal surface.