Ilex paraguariensis St. Hilaire (Aquifoliaceae) is a typical plant from South America. Preclinical studies have reported the effect of I. paraguariensis-based preparations on different alterations in the brain. This study aimed to examine the antidepressant-like and neuroprotective effects of I. paraguariensis hydroalcoholic extract (IpHE). The role of the N-methyl-D-aspartate receptor and the L-arginine-nitric oxide pathway in the IpHE antidepressant-like effect was also evaluated. Using the tail suspension test, we showed that IpHE (0.1-10 mg/kg, orally) exerts an antidepressant-like effect similar to that of ketamine (1 mg/kg, intraperitoneally). The antidepressant-like effect depends on the N-methyl-D-aspartate receptor and L-arginine-nitric oxide pathway modulation as we observed a combinatory effect using subeffective doses of IpHE (0.01 mg/kg, orally) and ketamine (0.1 mg/kg, intraperitoneally) or MK-801 (0.001 mg/kg, intraperitoneally). Also, pretreatment of mice with L-arginine (750 mg/kg, intraperitoneally) abolished the antidepressant-like effect of IpHE. This effect coincides with the neuroprotective effect, given that glutamate toxicity (10 mmol/l) did not decrease cell viability in hippocampal or cortical slices from IpHE-treated mice. The chromatographic profile of IpHE showed the presence of the methylxanthines caffeine and theobromine. Administration of methylxanthines (2.7 µg/kg) in mice produced an antidepressant-like effect, but not neuroprotection. We suggest that methylxanthines are at least in part responsible for the antidepressant-like effect of IpHE; further studies are necessary to determine the biological compounds responsible for the neuroprotective effect.