Gliomas are the most common type of malignant primary brain tumor and few risk factors have been linked to their development. Handedness has been associated with several pathologic neurological conditions such as schizophrenia, autism, and epilepsy, but few studies have evaluated a connection between handedness and risk of glioma. In this study, we examined the relationship between handedness and glioma risk in a large case–control study (1849 glioma cases and 1354 healthy controls) and a prospective cohort study (326,475 subjects with 375 incident gliomas). In the case–control study, we found a significant inverse association between left handedness and glioma risk, with left-handed persons exhibiting a 35% reduction in the risk of developing glioma [odds ratio (OR) = 0.65, 95% confidence interval (CI) 0.51–0.83] after adjustment for age, gender, race, education, and state of residence; similar inverse associations were observed for GBM (OR = 0.69, 95% CI 0.52–0.91), and non-GBM (OR = 0.59, 95% CI 0.42–0.82) subgroups. The association was consistent in both males and females, and across age strata, and was observed in both glioblastoma and in lower grade tumors. In the prospective cohort study, we found no association between handedness and glioma risk (hazards ratio = 0.92, 95% CI 0.67–1.28) adjusting for age, gender, and race. Further studies on this association may help to elucidate mechanisms of pathogenesis in glioma.