Bacterial pathogens can alter host cell mechanics to promote their own dissemination through tissues. Borrelia burgdorferi (Bb), the causative agent of Lyme Disease, is an extracellular bacterial pathogen that can spread to distant tissues by travelling in and through the vasculature, lined by a single continuous endothelial cell monolayer. To examine whether Bb infection induces changes in host endothelial cell biomechanics, we used video-microscopy to monitor endothelial cell responses to prolonged exposure to Bb. We found that initially Bb cells homogeneously spread over the endothelial cell monolayers maintaining their spirochete morphology and increased motility, but over time less motile Bb aggregates formed, some of which were intracellular, viable and persisted overtime. We hypothesized that the different forms that Bb attained over the course of infection (spirochetal versus aggregate form) could impact distinctly host cell biomechanics and response to infection. Consistently, we found that host cell motility, cell-extracellular matrix and cell-cell forces in response to infection were transiently reduced during the early stages of infection but reverted to levels similar to those of cells not exposed to infection at later stages when bacterial aggregates formed. To examine how biochemical signaling might regulate the time-dependent biomechanical responses to infection, we performed RNA sequencing and discovered upregulation of multiple host cell innate immune signaling pathways during early but not late infection where differences were minimal compared to cells originating from uninfected wells. Altogether our findings suggest that the changes in host cell innate immune signaling, and biomechanics are tightly regulated and linked to the different morphological forms that Bb cells attain. Our studies also shed light into the processes that may contribute in rendering Bb infection chronic and might facilitate efforts on better understanding Lyme disease.