Osteosarcoma is the most frequent type of primary bone cancer in children and adolescents. These malignant osteoid forming tumors are characterized by their uncontrolled hyperproliferation. Here, we investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the growth of human osteosarcoma. We show that alpha-CaMKII is expressed in human osteosarcoma cell lines and in primary osteosarcoma tissue derived from patients. The pharmacologic inhibition of CaMKII in MG-63 and 143B human osteosarcoma cells by KN-93 resulted in an 80 and 70% decrease in proliferation, respectively, and induced cell cycle arrest in the G(0)/G(1) phase. The in vivo administration of KN-93 to mice xenografted with human osteosarcoma cells significantly decreased intratibial and subcutaneous tumor growth. Mechanistically, KN-93 and alpha-CaMKII siRNA increased p21((CIP/KIP)) gene expression, protein levels, and decreased the phosphorylation of retinoblastoma protein and E2F transactivation. Furthermore, the inhibition of CaMKII decreased membrane-bound Tiam1 and GTP-bound Rac1, which are known to be involved in p21 expression and tumor growth in a variety of solid malignant neoplasms. Our results suggest that CaMKII plays a critical role in the growth of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat conventional high-grade osteosarcoma in children.