Affinin (spilanthol) is the main bioactive alkylamide present in roots, exerting antinociceptive and anti-inflammatory effects that involve the activation of TRP channels. Previous studies indicated that affinin reduces the LPS-induced increase in pro-inflammatory cytokine production in murine macrophages. However, no studies have evaluated whether affinin produces antinociceptive, anti-inflammatory, and behavioral effects in experimental animals treated with LPS, nor has the mechanism of action involved in these pharmacological effects been established. The present study evaluated whether affinin induces hypothermia, catalepsy, hypolocomotion, and analgesia and, moreover, whether the analgesia involves the activation of the CB1 cannabinoid receptor and TRPV1 and TRPA1 channels. Subsequently, the anti-inflammatory activity and behavioral effects induced by affinin (20 mg/kg) in mice were evaluated via LPS (2.5 mg/kg)-induced hypothermia. The results of the experiments indicate that the analgesic effect of affinin involves the activation of the CB1 cannabinoid receptors and the TRPV1 and TRPA1 channels. Additionally, affinin reduced the severity of LPS-induced hypothermia and attenuated the increase in TNF-α and IL-6 levels in serum. The results obtained demonstrate that affinin induces antinociceptive, anti-hypothermic, and anti-inflammatory activities, which involve the CB1 receptor and the TRPV1 and TRPA1 channels and the suppression of pro-inflammatory cytokines.