ETHNOPHARMACOLOGY RELEVANCE: Stachytarpheta cayennensis is a plant that is traditionally used to treat tegumentary leishmaniasis and as an anti-inflammatory agent. AIM OF THE STUDY: This study aimed to evaluate the action of S. cayennensis extracts on the Leishmania (Leishmania) amazonensis arginase enzyme. MATERIALS AND METHODS: S. cayennensis was collected from the Brazilian Amazon region. Aqueous extracts were fractionated with n-butanol. The leishmanicidal effects of the n-butanolic fraction (BUF) were evaluated in L. (L.) amazonensis promastigotes and amastigotes. BUF was tested against recombinant arginase from both L. (L.) amazonensis and macrophage arginase. Promastigote cultures and infected macrophage cultures were supplemented with L-ornithine to verify arginase inhibition. NMR analysis was used to identify the major components of BUF. RESULTS: BUF showed an EC of 51 and 32µg/mL against promastigotes and amastigotes of L. (L.) amazonensis, respectively. BUF contains a mixture of verbascoside and isoverbascoside (7:3 ratio) and is a potent L. (L.) amazonensis arginase inhibitor (IC=1.2µg/mL), while macrophage arginase was weakly inhibited (IC>1000µg/mL). The inhibition of arginase by BUF in promastigotes and amastigotes could be demonstrated by culture media supplementation with L-ornithine, a product of the hydrolysis of L-arginine by arginase. CONCLUSIONS: Leishmanicidal effects of the S. cayennensis BUF fraction on L. (L.) amazonensis are associated with selective parasite arginase inhibition.