A new cyclic depsipeptide, rakicidin F (1), along with the known compound rakicidin C (2), was isolated from the fermentation broth of the marine sponge-derived actinomycete strain Streptomyces sp. GKU 220. Their structures were elucidated by interpreting the HRFABMS and NMR spectroscopic data. Rakicidin F (1) showed growth inhibitory activity against Bacillus subtilis and Escherichia coli at the dosage of 25 μg per disk, while rakicidin C (2) showed weak activity only against B. subtilis with an MIC value of 50 μg per disk. Rakicidin C (2) treated at a dose of 1.25 μM achieved 30% inhibition of invading murine carcinoma colon 26-L5 cells without any cytotoxicity, and rakicidin F (1) did not show any anti-invasive effect at non-cytotoxic concentration. Rakicidin C (2) is less cytotoxic than rakicidin F (1) (IC50=17 μM). In conclusion, marine sponge-derived Streptomyces sp. GKU 220 produces a novel antibacterial cyclic depsipeptide rakicidin F (1) consisting of three amino acids, including a rare and nonproteinogenic amino acid (4-amino-2,4-pentadienoic acid), and a modified fatty acid. Rakicidins F (1) and C (2) have a different 3-hydroxy acid moiety from rakicidins A (3), B (4), D (5) and E (6), and a glutamine residue in place of the 3-hydroxyasparagine residue in 3, 4 and 5.