ABSTRACT Pyronaridine-artesunate efficacy for the treatment of uncomplicated Plasmodium falciparum malaria was assessed in an area of artemisinin resistance in western Cambodia. This nonrandomized, single-arm, observational study was conducted between 2014 and 2015. Eligible patients were adults or children with microscopically confirmed P. falciparum infection and fever. Patients received pyronaridine-artesunate once daily for 3 days, dosed according to body weight. The primary outcome was an adequate clinical and parasitological response (ACPR) on day 42, estimated by using Kaplan-Meier analysis, PCR adjusted to exclude reinfection. One hundred twenty-three patients were enrolled. Day 42 PCR-crude ACPRs were 87.2% (95% confidence interval [CI], 79.7 to 92.6%) for the overall study, 89.8% (95% CI, 78.8 to 95.3%) for Pursat, and 82.1% (95% CI, 68.4 to 90.2%) for Pailin. Day 42 PCR-adjusted ACPRs were 87.9% (95% CI, 80.6 to 93.2%) for the overall study, 89.8% (95% CI, 78.8 to 95.3%) for Pursat, and 84.0% (95% CI, 70.6 to 91.7%) for Pailin ( P = 0.353 by a log rank test). Day 28 PCR-crude and -adjusted ACPRs were 93.2% (95% CI, 82.9 to 97.4%) and 88.1% (95% CI, 75.3 to 94.5%) for Pursat and Pailin, respectively. A significantly lower proportion of patients achieved day 3 parasite clearance in Pailin (56.4% [95% CI, 43.9 to 69.6%]) than in Pursat (86.7% [95% CI, 76.8 to 93.8%]; P = 0.0019). Fever clearance was also extended at Pailin versus Pursat ( P < 0.0001). Most patients (95.9% [116/121]) harbored P. falciparum kelch13 C580Y mutant parasites. Pyronaridine-artesunate was well tolerated; mild increases in hepatic transaminase levels were consistent with data from previous reports. Pyronaridine-artesunate efficacy was below the World Health Organization-recommended threshold at day 42 for medicines with a long half-life (90%) for first-line treatment of P. falciparum malaria in western Cambodia despite high efficacy elsewhere in Asia and Africa. (This study has been registered at ClinicalTrials.gov under registration number NCT02389439.)