Variation in semicircular canal morphology correlates with locomotor agility among species of mammals. An experimental evolutionary mouse model was used to test the hypotheses that semicircular canal morphology (1) evolves in response to selective breeding for increased locomotor activity, (2) exhibits phenotypic plasticity in response to early-onset chronic exercise, and (3) is unique in individuals possessing the minimuscle phenotype. We examined responses in canal morphology to prolonged wheel access and selection in laboratory mice from four replicate lines bred for high voluntary wheel-running (HR) and four nonselected control (C) lines. Linear measurements and a suite of 3D landmarks were obtained from 3D reconstructions of μCT-scanned mouse crania (μCT is microcomputed tomography). Body mass was smaller in HR than C mice and was a significant predictor of both radius of curvature and 3D canal shape. Controlling for body mass, radius of curvature did not differ statistically between HR and C mice, but semicircular canal shape did. Neither chronic wheel access nor minimuscle affected radius of curvature or canal shape These findings suggest that semicircular canal morphology is responsive to evolutionary changes in locomotor behavior, but the pattern of response is potentially different in small- versus large-bodied species.