Chemotherapy treatment negatively affects the nervous and immune systems and alters gastrointestinal function and microbial composition. Outside of the cancer field, alterations in commensal bacteria and immune function have been implicated in behavioral deficits; however, the extent to which intestinal changes are related to chemotherapy-associated behavioral comorbidities is not yet known. Thus, this study identified concurrent changes in behavior, central and peripheral immune activation, colon histology, and bacterial community structure in mice treated with paclitaxel chemotherapy. In paclitaxel-treated mice, increased fatigue and decreased cognitive performance occurred in parallel with reduced microglia immunoreactivity, increased circulating chemokine expression (CXCL1), as well as transient increases in pro-inflammatory cytokine/chemokine (Il-1β, Tnfα, Il-6, and Cxcl1) gene expression in the brain. Furthermore, mice treated with paclitaxel had altered colonic bacterial community composition and increased crypt depth. Relative abundances of multiple bacterial taxa were associated with paclitaxel-induced increases in colon mass, spleen mass, and microglia activation. Although microbial community composition was not directly related to available brain or behavioral measures, structural differences in colonic tissue were strongly related to microglia activation in the dentate gyrus and the prefrontal cortex. These data indicate that the chemotherapeutic paclitaxel concurrently affects the gut microbiome, colonic tissue integrity, microglia activation, and fatigue in female mice, thus identifying a novel relationship between colonic tissue integrity and behavioral responses that is not often assessed in studies of the brain-gut-microbiota axis.