An unusual cyclic depsipeptide, pitiprolamide (1), was isolated from the marine cyanobacterium Lyngbya majuscula collected at Piti Bomb Holes, Guam. The structure was deduced using NMR, MS, X-ray crystallography, and enantioselective HPLC-MS techniques. Remarkably, proline represents half of the residues forming pitiprolamide (1). Other distinctive features include a 4-phenylvaline (dolaphenvaline, Dpv) moiety initially found in dolastatin 16 and the rare 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha) unit condensed in a unique sequence in one single molecule. Pitiprolamide (1) showed weak cytotoxic activity against HCT116 colon and MCF7 breast cancer cell lines, as well as weak antibacterial activities against Mycobacterium tuberculosis and Bacillus cereus.