We recently reported the identification and characterization of a novel small chemical molecule designated FL118. FL118 selectively inhibits multiple cancer survival and proliferation-associated antiapoptotic proteins (survivin, Mcl-1, XIAP, cIAP2) and eliminates small and large human tumor xenografts in animal models (Ling et al., PLoS One 2012, 7, e45571). Here, we report a follow-up study on the structure-activity relationship (SAR) of the hydroxyl group in the lactone ring of FL118. We found that the superior antitumor efficacy of FL118 heavily depends on its steric configuration through comparing the antitumor activity of FL118 with FL113 (the racemic mixture of FL118). Consistently, FL118 proved much more effective in inhibiting the expression of survivin, Mcl-1, and cIAP2, both in vitro and in vivo, compared to FL113. Additionally, Tet-on controlled induction of survivin or forced expression of Mcl-1 protects cancer cells from FL118-mediated growth inhibition and cell death. To further explore the SAR, we synthesized seven position 20-esterifiable FL118 and FL113 derivatives. Studies on these seven new compounds revealed that keeping a free hydroxyl group of FL118 is also important for high antitumor efficacy. Together, these studies confirm the superior anticancer activity of FL118 and narrow the window for further SAR studies to generate novel analogues based on FL118 core structure on its other potential chemical positions.