FW523-3, a new lipopeptide compound, was recently isolated and purified from the culture broth of a marine Micromonospora chalcea. FW523-3 was shown to inhibit the proliferation of certain cancer cells. However, the spectra and the underlying mechanism of its antitumor activity are unclear. In this study, the MTT and colony formation assays were employed to determine the antitumor spectra of FW523-3 and its effect on cell proliferation, respectively. Apoptosis was analyzed using DNA laddering assay and flow cytometry and the involved pathways were explored by Western blotting. Results revealed that FW523-3 exhibited cytotoxicity in a panel of tumor cell lines including esophageal squamous cell carcinoma cells (EC109), lung cancer cells (A549 and 95D), gastric cancer cells (SGC7901), uterine cervix cancer cells (HeLa) and hepatocellular carcinoma cells (HepG2). Based on these results, FW523-3 inhibited the colony formation ability of tumor cells. Moreover, FW523-3 induced apoptosis via activation of caspases 9, 7 and 3. FW523-3 also blocked the ERK and p38 signaling pathways. Taken together, we propose that FW523-3 acts as a broad-spectrum antitumor drug. FW523-3 inhibits tumor cell growth and induces tumor cell apoptosis via the mitochondrial and MAPK pathways.