Peumus boldus (P. boldus) is a medicinal plant popularly used in the treatment of gastrointestinal disorders. P. boldus aqueous extract is rich in phenolic compounds and alkaloids that possess antiinflammatory and antioxidant effects. In the present study, the potential protective effect of P. boldus against Cu²⁺-induced toxicity was investigated. Adult Drosophila melanogaster were exposed to Cu²⁺ (1mM and 3mM) and/or P. boldus aqueous extract (5mg/mL) in the food during 4days. Cu²⁺-fed flies had impairment in the negative geotaxis performance (i.e. motor climbing capability) as well as a higher incidence of mortality when compared to the control group. P. boldus co-treatment afforded protection against the Cu²⁺-induced toxicity. Acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity decreased significantly in D. melanogaster after Cu²⁺ exposure. P. boldus co-exposure for 4days restored enzyme activities to control levels. In addition, Cu²⁺ exposure caused a significant increase in the mRNA levels of antioxidant enzymes, superoxide dismutase (Sod1), catalase (Cat), thioredoxin reductase (TrxR1) and nuclear factor erythroid 2–related factor 2 (Nrf2), as well as increased the mRNA levels of acetylcholinesterase (Ace). The expression of P-type ATPase (Atp7A) and copper uptake protein 1 (Ctr1A) mRNAs were up-regulated in D. melanogaster exposed to Cu²⁺. The co-treatment with P. boldus blunted Cu²⁺-induced up-regulation of Atp7A and down-regulated Ctr1A mRNA expression. These findings suggest that P. boldus extracts reduce Cu²⁺-induced toxicity but not Cu²⁺ absorption in D. melanogaster. Consequently, P. boldus can be a potential therapeutical alternative for modulating Cu²⁺-associated toxicity.