Studies on host microbiota and their interactions with the central nervous system (CNS) have grown considerably in the last decade. Indeed, it has been widely demonstrated that dysregulations of the bidirectional gut–brain crosstalk are involved in the development of several pathological conditions, including chronic pain. In addition, the activation of central and peripheral glial cells is also implicated in the pathogenesis and progression of pain and other neurodegenerative disorders. Recent preclinical findings suggest that the gut microbiota plays a pivotal role in regulating glial maturation, morphology and function, possibly through the action of different microbial metabolites, including the most studied short-chain fatty acids (SCFAs). Moreover, altered microbiota composition has been reported in CNS disorders characterized by glial cell activation. In this review, we discuss recent studies showing the role of the gut microbiota and the effects of its depletion in modulating the morphology and function of glial cells (microglia and astrocytes), and we hypothesize a possible role for glia–microbiota interactions in the development and maintenance of chronic pain.