To evaluate the efficacy of mitogen-activated protein kinase/extracellular signal-related kinase kinase inhibitor PD-0325901 in advanced non-small cell lung cancer patients who had experienced treatment failure after, or were refractory to, standard systemic therapy. This open-label, phase II study initially evaluated 15 mg PD-0325901 twice daily administered intermittently (3 weeks on/1 week off; schedule A). As this schedule was not well tolerated, a second schedule was introduced as follows: 5 days on/2 days off for 3 weeks, followed by 1 week off (schedule B). The primary end point was objective response. All patients had received prior systemic therapy (median of two regimens, including epidermal growth factor receptor inhibitors in 26%). Of 13 patients treated on schedule A, three discontinued due to adverse events (blurred vision, fatigue, and hallucinations, respectively). Twenty-one patients received schedule B. Main toxicities included diarrhea, fatigue, rash, vomiting, nausea, and reversible visual disturbances. Hematologic toxicity consisted mainly of mild-to-moderate anemia, without neutropenia. Chemistry abnormalities were rare. Mean (coefficient of variation) PD-0325901 trough plasma concentrations were 100 ng/mL (52%) and 173 ng/mL (73%) for schedules A and B, respectively, above the minimum target concentration established in preclinical studies (16.5 ng/mL). There were no objective responses. Seven patients had stable disease. Median (95% confidence interval) progression-free survival was 1.8 months (1.5-1.9) and overall survival was 7.8 months (4.5-13.9). PD-0325901 did not meet its primary efficacy end point. Future studies should focus on PD-0325901 schedule, rational combination strategies, and enrichment of patient selection based on mode of action.