Abstract Body: Intestinal lymphatics are implicated in costly diseases such as obesity and Crohn’s disease, which impose a great risk for cardiovascular disease development. Lacteal lymphatic endothelial cell (LEC) discontinuous button junctions critical for interstitial fluid, immune cell, and lipid uptake can transform into continuous zipper junctions under infectious stimuli. Conversely, the mechanistic understanding of lacteal junctional regulation is sparse, precluding the development of novel therapeutics targeting intestinal lymphatics. VEGF-A/VEGFR2 signaling induces lacteal zippering through PI3K/AKT. FOXO1 is a direct downstream target of AKT; however, it is unknown if FOXO1 regulates lacteal junctions. To study this, we ablated Foxo1 before and after lacteal button development in LECs using a tamoxifen-inducible Flt4CreER^T2;Foxo1^flox/flox (Foxo1^Flt4iLEC-KO) mouse model to study lacteal button formation and transformation. Using our novel, semi-automated method on NIH ImageJ to analyze lacteal junctions, ablation of Foxo1 in lymphatics prevents lacteal button formation (n=3, p<0.05) and induces lacteal zippering after buttons develop (24.9% zipper increase). To investigate the mechanism of FOXO1 in lacteal junctional regulation, we cultured human dermal LECs (hdLECs) and performed FOXO1 knockdown in vitro. Our RNAseq data shows significant downregulation in the expression of cytoskeletal contraction protein and VE-cadherin kinase genes following FOXO1 knockdown (n=3, p<0.05), which also occurs at the protein level through preliminary western blot results. This suggests FOXO1 regulates lacteal LEC junctions through cytoskeletal contraction and VE-cadherin phosphorylation. To reverse lacteal zippering in vivo, we administered Dexamethasone to Foxo1^Flt4iLEC-KO mice and found lacteals remaining zippered (30.5% increase in zippers), suggesting FOXO1 contributes to Dexamethasone-induced lacteal button formation. We are determining if FOXO1 regulates the lacteal response to inflammation and found Indomethacin-induced bowel injury leading to lacteal zippering (12% increase in zipper junctions). We identify FOXO1 as a novel downstream effector in lacteal junctional regulation, rendering it a potential therapeutic target for diseases involving intestinal lymphatic junctions.