Natural depsipeptide vinylamycin was reported to be an antibiotic previously. Herein we report vinylamycin to be active against K562 leukemia cells (IC50 = 4.86 μM) and be unstable in plasma (t1/2 = 0.54 h). A total of 24 vinylamycin analogues with modification of the OH group and chiral centers were generated via a combinatorial approach. The lead compound 1a was subsequently characterized as having the following: no antimicrobial activity, significantly higher plasma stability (t1/2 = 14.3 h), improved activity against K562 leukemia cells (IC50 = 0.64 μM), and up to 75% cell inhibition without significant toxicities in K562 cells xenograft zebrafish model. Furthermore, compound 1a maintained its activity against the breast cancer cell line MCF-7 under hypoxic conditions. In comparison, the activity of gemcitabine in the same hypoxic in vitro model of MCF-7 cells was 15-fold lower. Therefore, the present results demonstrate that 1a has great potential as an anticancer agent.