Soil-transmitted intestinal helminth parasites infect billions of people worldwide, causing substantial morbidity and posing a significant global public health problem. Recent hallmark studies have shown that following helminth infection, specialized cells in the gut epithelium called tuft cells expand and trigger a hostprotective type 2 immune response that promotes parasite expulsion and tissue repair. Tuft cells are derived from stem cells at the base of the intestinal epithelial crypt, and transcription factors such as Pou2f3 that drive the maturation of tuft cells have been identified. However, despite these advances, the molecular mechanisms that control tuft cell abundance and anti-helminth function remain incompletely understood. Although it is established that microRNAs (miRNAs) as a class of regulatory molecules are critical for proper intestinal architecture and function, the roles of individual miRNAs in the intestinal epithelium are just now starting to emerge. We recently discovered that the whole-body genetic deletion of a single miRNA, miR-375, was associated with lower worm burdens after infection with Heligmosomoides polygyrus, a helminth parasite of mice used as a model for human hookworm infection. Moreover, the 375-/- mice exhibited increased abundance of tuft cells in the small intestine. Notably, we also determined through a bioinformatic screen that Pou2f3 is a predicted target of miR-375. This proposal is focused on bridging two critical knowledge gaps: (1) Is the effect on H. polygyrus worm burden mediated by loss of miR-375 function in the intestinal epithelial cell (IEC) lineage, immune cell lineage, or both? and (2) Does the effect of miR-375 loss depend on Pou2f3-mediated maturation of tuft cells? In the first Aim, we will define the impact of IEC-specific miR-375 loss on resistance to H. polygyrus. To accomplish this goal, we will apply two independent, complementary strategies. Specifically, we will: (i) generate bone marrow chimeric mice, in which miR-375 deficiency is restricted to non-immune cells, and also (ii) breed miR-375fl/fl;Vil1-Cre mice, in which miR-375 deficiency is specific to IECs, and compare H. polygyrus worm burden after infection with what we have observed previously in whole-body miR-375-/- mice. In the second Aim, we will establish whether tuft cells are required for the effects of miR-375 loss on H. polygyrus worm burden. We will first determine whether Pou2f3 is a direct target of miR-375 and also leverage single cell transcriptomics to identify additional candidate miR-375 target genes whose expression levels are significantly increased in intestinal epithelial stem and/or secretory progenitor cells of 375-/- mice during the response to helminth infection. We will then perform genetic studies to determine whether loss of Pou2f3 is sufficient to negate the positive effects of miR-375 loss on H. polygyrus worm burden. The completion of these studies will substantially advance the field by establishing that an IEC miRNA regulates tuft cell-mediated anti-helminth function during H. polygyrus infection. Improved understanding of the key epithelial regulators of the host response to helminth will be critical for developing new strategies to treat and prevent infection.