In addition to having anti-inflammatory properties, non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neoplastic cell proliferation by inducing apoptosis. Inhibition of cyclooxygenase-2 (COX-2) seemed to be the principal target of NSAIDs, as it is overexpressed in several cancers and catalyzes the synthesis of prostaglandin E2 (PGE2), the critical pro-inflammatory molecule. A major role for phosphatidylinositol-3 kinase (PI3-kinase) pathway activation in human tumors has been more recently established. The present study explored the role of PI3-kinase and Wnt molecular pathways in COX-2 and PGE2 production as well as NSAIDs' chemopreventive effect in colon cancer. 1,2-dimethylhydrazine (DMH) was used for experimental colon cancer model in rat and diclofenac as the preferential COX-2 selective chemopreventive agent. Expression of caspase-3 and caspase-9 was checked in the colonic tissue by immunofluorescence. A decrease was seen in their expressions, indicative of inhibition of apoptosis in the present model. COX-2 mRNA expression as well as PGE2 levels was elevated after DMH treatment; however, COX-1 mRNA expression was unaltered as seen by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. DMH also activated PI3-kinase, Akt, Wnt, and β-catenin expressions but reduced the glycogen synthase kinase-3β (GSK-3β) levels. Co-administration of diclofenac with DMH increased the mRNA expression of GSK-3β while inactivating PI3-kinase, Akt, Wnt, and β-catenin. The study suggests that activation of PI3-kinase and Wnt signaling is associated with COX-2/PGE2 production and in turn inhibition of apoptosis in colon cancer, while diclofenac targeted these pathways to restore apoptosis in the present system.