A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser473 is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A2α (cPLA2α) on AKT phosphorylation at Ser473 and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser473 was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CAE545K mutation) and HT-29 (PIK3CAP499T mutation). Over-expression of cPLA2α by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD-1 cells. In contrast, silencing of cPLA2α with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by >90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA2α expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA2α is required for sustaining AKT phosphorylation at Ser473 and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy.