Abstract The intestinal epithelium plays a central role in controlling host-microbe interactions by acting as a physical barrier, a sensor of luminal microorganisms, and a signal transducer of microbial stimulation. This function is largely mediated by Toll-like receptor (TLR) signaling in the intestinal epithelial cells. Increasing evidence indicate that TLR-mediated host-microbe interactions are critical for shaping the host immune system and maintaining intestinal homeostasis. As such, epithelial TLR signaling must be precisely regulated to prevent diseases such as necrotizing enterocolitis, autoimmune disorders, inflammatory diseases, and cancer. Thus, studying mechanisms underlying epithelial TLR signaling regulation is of great importance. Our previous studies reveal that hnRNP I, an RNA-binding protein, is a novel regulator of neonatal intestinal epithelial innate immunity through downregulating IRAK1-mediated TLR signaling. Inspired by this observation, we went on to determine if hnRNP I regulates intestinal epithelial TLR signaling in adulthood, a stage when intestinal epithelial cells have acquired immune tolerance to commensal microbes. We found that loss of hnRNP I in adult intestinal epithelial cells results in severe crypt cell apoptosis and loss of intestinal stem cells, highly resembling defects of TLR4 activation in crypt cells. Strikingly, nuclear accumulation of IRAK1, a key component of TLR4 signaling, is dramatically reduced in the crypt cells. Based on these exciting findings, we formulated the hypothesis that hnRNP I regulates IRAK1-dependent TLR4 signaling in crypt cells to guard crosstalk between crypt cells and luminal microbes. As initial steps to test this hypothesis, we propose to investigate if hnRNP I regulates crypt cell innate immunity through the control of TLR4-mediated signaling. Moreover, we propose to investigate if loss of hnRNP I reduces the immune tolerance of crypt cells to commensal microbes. Completion of the proposed studies will build a solid foundation for our future in-depth mechanistic studies on hnRNP I-dependent TLR signaling regulation, which will likely uncover novel mechanisms important for the control of innate immunity in the intestinal stem cell compartments.