Prostate cancer patients with regional lymph node involvement at radical prostatectomy often experience disease progression to other organs, with the bone as the predominant site. The transcription factor Runx2 plays an important role in bone formation and prostate cancer cell migration, invasion, and metastasis. Here we showed that the forkhead box O (FOXO1) protein, a key downstream effector of the tumor suppressor PTEN, inhibits the transcriptional activity of Runx2 in prostate cancer cells. This inhibition was enhanced by PTEN but diminished by active Akt. FOXO1 bound to Runx2 in vitro and in vivo and suppressed Runx2's activity independent of its transcriptional function. FOXO1 inhibited Runx2-promoted migration of prostate cancer cells, whereas silencing of endogenous FOXO1 enhanced prostate cancer cell migration in a Runx2-dependent manner. Forced expression of FOXO1 also inhibited Runx2-promoted prostate cancer cell invasion. Finally, we found that expression of PTEN and the level of FOXO1 in the nucleus is inversely correlated with expression of Runx2 in a cohort of prostate cancer specimens from patients with lymph node and bone metastasis. These data reveal FOXO1 as a critical negative regulator of Runx2 in prostate cancer cells. Inactivation of FOXO1 due to frequent loss of PTEN in prostate cancer cells may leave the oncogenic activities of Runx2 unchecked, thereby driving promiscuous expression of Runx2 target genes involved in cell migration and invasion and favoring prostate cancer progression.