The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5(K_D= 9.48 nmol/L for Bcl-2) and SM-6(K_D= 0.08 nmol/L for Bcl-xL), which exhibited improved binding affinity compared with the lead Bim(K_D= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively). These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core. The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2 family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and / The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Interestingly the analogue SM-5 (K_D = 9.48 nmol / L for Bcl-2) and SM-6 (K_D = 0.08 nmol / L for Bcl-xL), which developed improved binding affinity compared with the lead Bim nmol / L for Bcl-2 and 22.2 nmol / L for Bcl-xL, respectively). These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.