The objective of this study was to evaluate the antitumor activity and safety profile of 5 mg/m2 plitidepsin administered as a 3-h continuous intravenous infusion every 2 weeks to patients with advanced malignant melanoma who relapsed or progressed after one line of systemic therapy. Objective response rate (primary efficacy endpoint) was evaluated according to Response Evaluation Criteria In Solid Tumors and toxicity was assessed using National Cancer Institute -Common Toxicity Criteria Version 2.0. Of 39 enrolled patients (median age: 53 years), 37 patients were treated who received a total of 167 treatment cycles (median: 3 cycles per patient; range: 1–32). All patients had received prior systemic therapy with a median of one line per patient (range: 1–6 lines). Of the 35 evaluable patients, two dacarbazine-resistant patients (5.7%) with metastatic cutaneous melanoma achieved partial responses. Five other patients (14.3%) reported stable disease (median stable disease duration: 3.5 months; range: 2.2–15.8 months). Therefore, the rate of tumor growth control was 20.0%. With a median follow-up of 11.0 months, the median progression-free survival was 1.3 months and the median overall survival was 3.5 months. Six patients (16.2%) had the following treatment-related grade 3/4 adverse events: myalgia (n = 3), injection-site reaction (n = 2), hypersensitivity, hypotension, and fatigue (n = 1 each). One patient was withdrawn from the trial because of grade 4 hypersensitivity reaction and hypotension. No severe neutropenia was reported. Plitidepsin showed a minor degree of antitumor activity in patients with refractory advanced malignant melanoma. Further evaluation of plitidepsin in combination schedules may be warranted.