Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.