Purpose: A dozen clinical trials examining a combination of temsirolimus and cetuximab in treating metastatic colon cancer are currently underway. We investigated the role of cancerous inhibitor of protein phosphatase 2A (CIP2A) in the synergism between temsirolimus and cetuximab in colon cancer. Methods: Five colon cancer cell lines were used for in vitro studies. Signal transduction pathways were assessed by immunoblotting. The synergism between studied drugs was analyzed with combination indexes. Gene silencing was performed using small interfering RNAs. The efficacies of temsirolimus and cetuximab were tested in nude mice with colon cancer xenografts. Transcriptional activity was assessed using a reporter assay. The inhibitors leupeptin, chloroquine, and MG132 were used to assess protein degradation. The association between CIP2A, clinicopathological parameters, and survival was examined by immunohistochemical staining using a tumor tissue microarray. Results: Temsirolimus decreased the resistance of cells to cetuximab by both inhibiting transcription of CIP2A and increasing degradation of CIP2A through the lysosomal-autophagy pathway. The mammalian target of rapamycin (mTOR) protein immunoprecipitated along with CIP2A. Temsirolimus decreased expression of phosphorylated extracellular regulated protein kinase (pErk) and phosphorylated v-akt murine thymoma viral oncogene (pAKT) and decreased the interaction of CIP2A and mTOR in cell lines without the K-ras codon 12 mutation. CIP2A was a prognostic marker only in colon cancer patients with weak expression of pErk or pAKT. Conclusions: Temsirolimus decreases cellular resistance to cetuximab by regulating CIP2A expression in colon cancer cells. Potential biomarkers for CIP2A inhibitors include pErk and pAKT.