Background: In previous studies, the Forkhead/winged-helix-box-class-O3 (FOXO3) transcription factor has displayed both tumour suppressive and metastasis-promoting properties. To clarify its role in human colorectal cancer (CRC) progression, we examined in vivo FOXO3 expression at key points of the metastatic cascade. Methods: Formalin-fixed paraffin-embedded resection specimens from normal colon, adenomas, primary CRC specimens of different pathological stage and CRC specimens with matched liver metastases were used to generate three separate custom-designed tissue microarray (TMA) representations of metastatic progression. Triplicate cores, immunostained for FOXO3 were scored semiquantitatively by two investigators. Results: The FOXO3 expression is significantly reduced in CRC specimens compared with normal tissue, and progressive FOXO3 downregulation is associated with advancing pathological stage. In addition, recurrent stage I/II primary tumours show a significantly lower FOXO3 expression compared with stage-matched non-recurrent tumours. When stratified according to high and low FOXO3 expression, mean disease-free survival in the low-expressing group was 28 months (95% CI 15.8–50.6) compared with 64 months (95% CI 52.9–75.4) in the high-expressing group. Conclusion: We have demonstrated an association between low FOXO3 expression and CRC progression in vivo using purpose-designed TMAs. Forkhead/winged-helix-box-class-O3 may represent a novel biomarker of nodal and distant disease spread with clinical utility in CRC.