Arthopods such as Ixodes scapularis ticks serve as vectors for many human pathogens. The arthropod gut presents a pivotal microbial entry point and determines pathogen colonization and survival. We show that the gut microbiota of I. scapularis, a major vector of the Lyme disease spirochete Borrelia burgdorferi, influence spirochete colonization of ticks. Perturbing the gut microbiota of larval ticks reduced Borrelia colonization, and dysbiosed larvae displayed decreased expression of the transcription factor signal transducer and activator of transcription (STAT). Diminished STAT expression corresponded to lower expression of peritrophin, a key glycoprotein scaffold of the glycan-rich mucus-like peritrophic matrix (PM) that separates the gut lumen from the epithelium. The integrity of the I. scapularis PM was essential for B. burgdorferi to efficiently colonize the gut epithelium. These data elucidate a functional link between the gut microbiota, STAT-signaling, and pathogen colonization in the context of the gut epithelial barrier of an arthropod vector. Display omitted •Tick gut microbiota modulate Borrelia burgdorferi colonization of the gut•Gut microbiota maintain the integrity of the peritrophic membrane of the tick gut•The integrity of the peritrophic membrane promotes Borrelia colonization The tick Ixodes scapularis is a major vector of the Lyme disease spirochete Borrelia burgdorferi. Narasimhan et al. find that tick gut microbiota affect spirochete colonization by regulating the integrity of the peritrophic matrix, a mucus-like lining of the arthropod gut, via transcription factor STAT-mediated control of peritrophin expression.