Background: The purpose of this study was to evaluate the expression of Notch-induced transcription factors (NTFs) HEY1, HES1 and SOX9 in colorectal cancer (CRC) patients to determine their clinicopathologic and prognostic significance. Methods: Levels of HEY1, HES1 and SOX9 protein were measured by immunohistochemistry in a nonmalignant and malignant tissue microarray of 441 CRC patients, and the findings correlated with pathologic, molecular and clinical variables. Results: The NTFs HEY1, HES1 and SOX9 were overexpressed in tumours relative to colonic mucosa (OR=3.44, P<0.0001; OR=7.40, P<0.0001; OR=4.08 P<0.0001, respectively). HEY1 overexpression was a negative prognostic factor for all CRC patients (HR=1.29, P=0.023) and strongly correlated with perineural and vascular invasion and lymph node (LN) metastasis. In 5-fluorouracil (5-FU)-treated patients, the tumour overexpression of SOX9 correlated with markedly poorer survival (HR=8.72, P=0.034), but had no predictive effect in untreated patients (HR=0.70, P=0.29). When HEY1, HES1 and SOX9 expression were combined to predict survival with chemotherapy, in treated patients there was an additive increase in the risk of death with each NTF overexpressed (HR=2.09, P=0.01), but no prognostic import in the untreated patient group (HR=0.74, P=0.19). Conclusion: The present study is the first to discover that HEY1 overexpression correlates with poorer outcome in CRC, and NTF expression is predictive of CRC patient survival with 5-FU chemotherapy. If confirmed in future studies, testing of NTF expression has the potential to enter routine pathological practice for the selection of patients to undergo chemotherapy alone or in combination with Notch inhibitors.