The halogenase gene spmH was putatively involved in the biosynthesis of spiroindimicins/indimicins (SPMs/IDMs), a group of chlorinated tryptophan dimers (TDs) from deep-sea-derived Streptomyces sp. SCSIO 03032. Inactivation of spmH led to six deschloro-analogues of TDs, including four new compounds SPMs G (1) and H (2), and IDMs F (3) and G (4). The structures and absolute configurations of 1-4 were unambiguously determined by the combination of extensive spectroscopic analysis, single-crystal X-ray diffraction and quantum chemical ECD calculations. Compounds 1 and 2 exhibited moderate cytotoxic activities against four cancer cell lines. Additionally, SpmH was biochemically characterized in vitro as an l-tryptophan 5-halogenase.