Colonization of a localized area of human skin byBorrelia burgdorferiafter a bite from an infected tick is the first step in the development of Lyme disease. The initial interaction between the pathogen and the human host cells is suggested to impact later outcomes of the infection. MicroRNAs (miRNAs) are well known to be important regulators of host inflammatory and immune responses. While miRNAs have been shown to play a role in the inflammatory response toB.burgdorferiat late stages of infection in the joints, the contributions of miRNAs to earlyB.burgdorferiinfection have yet to be explored. To address this knowledge gap, we used the published host transcriptional responses toB.burgdorferiin erythema migrans skin lesions of early Lyme disease patients and a human dermal fibroblasts (HDFs)/B.burgdorferico-culture model to predict putative upstream regulator miRNAs. This analysis predicted a role for miR146a-5p in both,B.burgdorferi-infected skin and -stimulated HDFs. miR146a-5p was confirmed to be significantly upregulated in HDF stimulated withB.burgdorferifor 24 hours compared to uninfected control cells. Furthermore, manipulation of miR146a-5p expression (overexpression or inhibition) altered theB.burgdorferidriven inflammatory profile of HDF cells. Our results suggest that miR146a-5p is an important upstream regulator of the transcriptional and immune early response to earlyB.burgdorferiinfection.