Abstract Langerhans cells (LCs), the skin residing dendritic cells (DCs), control both the induction of adaptive immunity, and immune tolerance in skin and are involved in variety of skin disease development. However, the regulatory mechanisms involved in the development and functions of LCs have not been completely elucidated. MicroRNAs (miRNAs), a class of non-coding small RNAs, are recognized as important regulators of protein-coding genes through the inhibition of mRNA translation. Using Cre-loxP Dicer deletion mouse models, our laboratory and others have reported that deletion of miRNAs by CD11c-Cre or hLangerin-Cre significantly reduced the number and interrupted the function of LCs, indicating that miRNAs are required for LC homeostasis and function after birth. While there are more than 1000 experimentally reported miRNAs, very few individual miRNAs are linked to LCs so far. We were the first to report that miR-150 and miR-223 differentially regulated LC-induced T cell proliferation and cytokine production. Most recently, our embryonic lineage-tracing studies showed that miRNAs, including miR-17-92 cluster, regulate LC embryonic development. Furthermore, using miRNA arrays, we identified that mature LCs have a unique miRNA gene expression profile compared to immature LCs, and that miRNA expression is dynamically changed during LC embryonic ontogeny. These findings led to our central hypothesis that the dynamically changed miRNAs may serve as critical regulators controlling LC ontogeny, homeostasis and function through fine-tuning specific target genes. In Aim 1, we will investigate the roles of miRNAs in LC ontogeny and homeostasis. Constitutive or inducible Csf1r-specific individual miRNA mutant mice will be used for studying embryonic LC ontogeny and LC repopulation after inflammation, while LC-specific Dicer or individual miRNA mutant mice will be used for LC homeostasis after birth. In Aim 2, we will investigate the roles of miRNAs in LC function, inducible LC-specific Dicer or individual miRNA mutation mouse models will be used. In Aim 3, the direct target gene(s) of miRNAs and related signaling pathways involved in LC development and function will be investigated by the combination of RNA-seq, miRNA bioinformatics and related target functional validation strategies. The proposed studies will uncover the dynamic miRNA-mRNA regulation and related molecular mechanisms and signaling pathways that control LC development and function, which will not only provide new insight into the biology of LCs, but may also facilitate the development of LC-based intervention strategies for diseases.