The tea of aerial parts of Smilax larvata Griseb. (Smilacaceae) has been ethnopharmacologically used in Southern Brazil due to its anti-inflammatory action. In this study, ethanolic and organic extracts from aerial parts of S. larvata were phytochemically and pharmacologically characterized. The phytochemical analysis of EtOAc extract of S. larvata revealed the presence of three flavonoids, drabanemoroside, kaempferol 3-O-α-L-rhamnopyranosyl(1→2)-α-L-rhamnopyranoside, and kaempferol, the first two being isolated for the first time in this genus, two phenolic compounds p-hydroxybenzoic acid and p-coumaric acid, and alkaloids. In vitro assays demonstrated a potential antioxidant property of SLG. The treatment with SLG induced a significant reduction of the formalin-evoked flinches in rats, an effect reversed by opioid antagonist naloxone. Treatment with SLG also induced a significant increase in the hot plate latency and a decrease of intestinal motility by 45%. No effect was observed over nociceptive responses induced by a TRPA1 agonist mustard oil or over acetic acid-induced writhing in mice. Together, our data suggested that SLG has an in vivo antinociceptive effect, which seems to be associated with the opioid system activation. These findings support previous claims of medical use of Smilax larvata in the treatment of pain conditions.