This study examined the role of glutamatergic system in the ascorbic acid (AA)-induced antinociception in chemical behavioural models of nociception in mice. AA (0.3–10 mg/kg, i.p.) produced significant inhibition of both phases of formalin-induced licking, with mean ID50 values of 4.0 and 3.2 mg/kg and inhibitions of 56 ± 4 and 60 ± 7% for the early and second phase of the nociception caused by formalin, respectively. AA (1–5 mg/kg, i.p.) also produced significant inhibition of glutamate-induced nociception with mean ID50 value of 2.1 mg/kg and inhibition of 66 ± 5%. Furthermore, AA (3 mg/kg, i.p.) caused marked inhibition of nociceptive response induced by intrathecal injection of glutamate, NMDA, AMPA, kainate and substance P, with inhibitions of 49 ± 9, 42 ± 7, 34 ± 8, 38 ± 5 and 65 ± 8%, respectively. In contrast, AA at the same dose did not affect the biting response induced by the metabotropic agonist trans-ACPD. Taken together, present results indicate that AA, at low systemic doses, produces a rapid onset and consistent antinociception in mice when assessed in several models of chemical nociception, an action that is likely mediated by an interaction with ionotropic, but not metabotropic, glutamate receptors.