Background: The present study was designed to evaluate the anti-hyperalgesic effect of kaempferol-3,7-di-O-alpha-L-rhamnopyranoside isolated from the ethyl acetate soluble part of Dryopteris cycadina. Pretreatment of the compound at the doses of 2.5, 5, and 10 mg/kg caused a significant reduction in abdominal constrictions in acetic acid-induced writhing test with maximum effect of 63.03% (P < 0.001) at 10 mg/kg i. p. When subjected in formalin test, it evoked a marked antinociceptive effect in both phases in a dose-dependent manner. The maximum (p < 0.01) pain-inhibiting effects were 61.36% and 65.89% in 1 st and 2 nd phases at 10 mg/kg i. p., respectively. Administration of atropine (non-selective cholinergic receptor antagonist) significantly (p < 0.05) antagonized the antihyperalgesic effect of the compound, while glibenclamide and naloxone did not alter the induced antinociceptive effect and thus, antinociceptive activity of the compound is mediated, at least in part, through cholinergic system antagonism; independent of calcium channel and opioidergic receptor participation. Furthermore, docking studies underlined strong COX-2 inhibitory activity of the compound.