A dense glycocalyx, composed of the megaDalton-sized membrane mucin MUC17, coats the microvilli in the apical brush border of transporting intestinal epithelial cells, called enterocytes. The formation of the MUC17-based glycocalyx in the mouse small intestine occurs at the critical suckling-weaning transition. The glycocalyx extends 1 µm into the intestinal lumen and prevents the gut bacteria from directly attaching to the enterocytes. To date, the mechanism behind the positioning of MUC17 to the brush border is not known. Here, we show that the actin-based motor proteins MYO1B and MYO5B, and the sorting nexin SNX27, regulate apical targeting of MUC17 in enterocytes. We demonstrate that MUC17 turnover at the brush border is slow and controlled by MYO1B and SNX27. Furthermore, we report that MYO1B regulates MUC17 protein levels in enterocytes, whereas MYO5B specifically governs MUC17 levels at the brush border. Together, our results extend our understanding of the apical targeting of membrane mucins and provide mechanistic insights into how defective positioning of MUC17 renders enterocytes sensitive to bacterial challenges.