This study investigated the role of the glutamatergic system on the antinociception caused by Polygala sabulosa hydroalcoholic extract (HE). The systems mediated by substance P, capsaicin, interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) were also investigated. P. sabulosa HE given orally produced a significant inhibition of glutamate-induced paw licking [ID(50) = 530.3 (416.7-674.8) mg/kg and inhibition of 79 +/- 6% at 1000 mg/kg]. The plant derivatives alpha-spinasterol, scopoletin and styryl-2-pyrones (compound 1 and 3) (10 mg/kg, intraperitoneally) inhibited 80 +/- 7%, 46 +/- 11%, 45 +/- 11% and 35 +/- 13% the nociceptive response caused by glutamate, respectively. Furthermore, P. sabulosa HE (500 mg/kg, orally) caused marked inhibition of nociceptive response induced by intrathecal injection of glutamate, N-methyl-d-aspartic acid, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, TNF-alpha and IL-1beta, with inhibitions of 44 +/- 7%, 55 +/- 4%, 38 +/- 10%, 61 +/- 7%, 76 +/- 9% and 100%, respectively. In contrast, P. sabulosa HE (500 mg/kg, orally) did not affect the biting response induced by the metabotropic glutamatergic receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, substance P and capsaicin. The locomotor activity was altered only in mice treated with a very high dose (1000 mg/kg) of P. sabulosa HE. Our results showed that the antinociceptive effects of P. sabulosa HE are associated with an inhibition of glutamatergic transmission and an inhibition of pathways dependent on pro-inflammatory cytokines. The plant derivatives alpha-spinasterol, scopoletin and styryl-2-pyrones play an important role on the antinociceptive effects of P. sabulosa HE.