STRADalpha is a pseudokinase that forms a heterotrimeric complex with the scaffolding protein MO25 and the tumor suppressor serine threonine protein kinase LKB1. Mutations in the LKB1 gene are responsible for the Peutz-Jeghers Syndrome (PJS) characterized by a predisposition to hamartomatous polyps and hyperpigmentation of the buccal mucosa. Mutations in LKB1 have also been observed in some sporadic tumours unrelated to PJS. The LKB1/STRAD/MO25 complex is involved in the regulation of numerous signaling pathways including metabolism, proliferation and cellular polarity of human intestinal epithelial cells. Cell polarization, together with tissue-restricted transcription, represents the main feature of enterocyte differentiation. Since a full-length STRADalpha transcript has not been identified thus far in these cells, we evaluated the expression of endogenous STRADalpha in five colorectal cancer cell lines characterized by their diverse ability to differentiate in vitro. We report herein the discovery of several novel splice isoforms of STRADalpha that differentially affect the kinase activity, complex assembly, subcellular localization of LKB1 and the activation of the LKB1-dependent AMPK pathway.