Nm23-H1 was the first metastasis suppressor discovered in most tumor models and reduction or loss of nm23-H1 expression correlates with tumor progression and metastasis in non-small-cell lung cancer. Despite extensive studies, the regulatory mechanism of nm23-H1 expression is far from elucidated. The transcriptional factor forkhead box (FOX)O3 has been reported to be involved in multiple regulatory signaling pathways in the biological behavior of tumors. Therefore, we aimed to study the relationship between FOXO3 activity and nm23-H1 expression. Real time reverse transcriptase-polymerase chain reaction and Western blotting assays were employed to determine nm23-H1 messenger ribonucleic acid and protein expression after being transformed by different FOXO3 plasmid in A549 cells. A dual luciferase reporter system and chromatin immunoprecipitation assay, were used to determine the promoter activity of the nm23-H1 gene and to detect the binding of FOXO3 into the nm23-H1 promoter, respectively. We found that activated FOXO3 decreased nm23-H1 expression and dominant negative FOXO3 increased nm23-H1 expression. Modulation of FOXO3 activity with FOXO3 pathway inhibitors altered nm23-H1 promoter activity. Although there is a putative binding site of FOXO3 in the nm23-H1 promoter, FOXO3 regulated nm23-H1 expression in an indirect manner. We demonstrated that the transcriptional factor FOXO3 decreased the expression levels of the tumor suppressor gene nm23-H1 in the non-small-cell lung cancer A549 cell line and that the level of expression of nm23-H1 was controlled by FOXO3 in an indirect manner. This finding provided an insight into the upstream regulation of nm23-H1 and may provide promising targets for inhibition of the metastasis process.