Tallysomycin (TLM) H-1, a novel TLM analog, is the major product isolated from Streptoalloteichus hindustanus SB8005, a genetically engineered strain from S. hindustanus E465-94 ATCC 31158. Based on the structural comparison and experimental assays, TLM H-1 represents a novel bleomycin (BLM) analog displaying DNA cleavage activity similar to its parent compounds TLM and BLM, both representatives of the glycopeptide anticancer antibiotics. The low titer of TLM H-1 in the engineered SB8005 strain has greatly limited its further study. In this paper, fermentation optimization for TLM H-1 production in the SB8005 strain is described; single-factor optimization and response surface methodology proved invaluable. The results indicated that three variables including distiller's grains and solubles, copper sulfate, and maltose out of eight parameters could significantly influence the TLM H-1 production. With systematic comparison and evaluation, the final optimized fermentation medium was determined. The optimized yield of TLM H-1 in the bench-top fermentor was 249.9 mg/L, which is 26.8 times higher than reported using the original medium, and 12.9-fold higher than that of the parent compound TLM produced by the wild-type strain. This work provides important parameters for TLM H-1 production by fermentation and should facilitate further mechanistic studies and clinical developments of TLM H-1 as an anticancer agent.