Purpose: Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. Experimental Design: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14þ cells. GMP-grade peptide was further validated for activity. Results: We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. Conclusions: These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.