Background: The efficacy of ipilimumab as a single agent and in combination for the treatment of melanoma and other malignancies has validated the immune checkpoints T-lymphocyte-associated protein 4 (CTLA-4) blockade in immunotherapy. Clinical trials that include anti-CTLA-4 in a combination design have gained traction. However, ipilimumab is the only anti-CTLA-4 antibody therapeutic approved worldwide. Therefore, novel anti-CTLA-4 therapeutics that may overcome the shortcomings of ipilimumab to fulfill the unmet medical needs is desperately desired. Methods: Mouse hybridoma cells were screened, cloned, and lead antibodies were humanized to obtain candidates with high activity in blocking CTLA-4/CD80 interaction. The resulting lead antibody was further characterized with in vitro binding and blocking assays, as well as mixed lymphocyte reaction assay. A humanized CTLA-4-B6 mouse model was used to evaluate the in vivo tumor growth inhibition activity of the antibody. Results: CBT-509, a novel IgG1 humanized monoclonal antibody against CTLA-4 was obtained. EC50 of CBT-509 binding with CTLA-4 and IC50 of CBT-509 blocking CTLA4/CD80 interaction were comparable to ipilimumab. CBT-509 binds to a distinct epitope, and has a high affinity with CTLA-4 (KD = 2.73E-10 M), more than 4-fold stronger than ipilimumab (KD = 1.21E-9 M). Compared with ipilimumab, CBT-509 was more active in mixed lymphocyte reaction, suggesting that it is more potent in human T-cell activation. Importantly, the efficacy of CBT-509 in killing tumor cells in vivo is much stronger than that of ipilimumab. In a MC38 murine colorectal cancer model under huCTLA-4 B6 mice background, CBT-509 eradicated tumors in 7 of 8 mice at a dose of 3 mg/kg, while ipilimumab eradicated tumors in 4 of 8 mice at the same dosage. Conclusions: Taken together, CBT-509 demonstrated an improvement over ipilimumab. Future efforts will focus on developing molecules that can further improve the efficacy and safety of anti-CTLA-4 based therapeutics.