Because Notch signaling is implicated in colon cancer tumorigenesis and protects from apoptosis by inducing pro-survival targets, it was hypothesized that inhibition of Notch signaling with gamma-secretase inhibitors (GSIs) may enhance the chemosensitivity of colon cancer cells. We first show that the Notch-1 receptor and its downstream target Hes-1 is upregulated with colon cancer progression, similar to other genes involved in chemoresistance. We then report that chemotherapy induces Notch-1, as oxaliplatin, fluorouracil (5-FU), or SN-38 (the active metabolite of irinotecan), induced Notch-1 Intracellular Domain (NICD) protein and activated Hes-1. Induction of NICD was caused by an increase in the gamma-secretase protein subunits, nicastrin and presenilin-1, as suppression of nicastrin with small interfering RNA (siRNA) prevented NICD induction after oxaliplatin. Subsequent, inhibition of Notch-1 signaling with a sulfonamide GSI (GSI34) prevented the induction of NICD by chemotherapy and blunted Hes-1 activation. Blocking the activation of Notch signaling with GSI34 sensitized cells to chemotherapy and was synergistic with oxaliplatin, 5-FU, and SN-38. This chemosensitization was mediated by Notch-1, as inhibition of Notch-1 with siRNA, enhanced chemosensitivity whereas overexpression of NICD increased chemoresistance. Downregulation of Notch signaling also prevented the induction of pro-survival pathways, most notably PI3K/Akt, after oxaliplatin. In summary, colon cancer cells may upregulate Notch-1 as a protective mechanism in response to chemotherapy. Therefore, combining GSIs with chemotherapy may represent a novel approach for treating metastatic colon cancers by mitigating the development of chemoresistance.