The tumor suppressor PTEN is a key regulator of a plethora of cellular processes that are crucial in cancer development. Through its lipid phosphatase activity PTEN suppresses the PI3K/AKT pathway to govern cell proliferation, growth, migration, energy metabolism and death. The repertoire of roles fulfilled by PTEN has recently been expanded to include crucial functions in the nucleus, where it favors genomic stability and restrains cell cycle progression, as well as protein phosphatase dependent activity at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), where PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and regulates Ca(2+) release from the ER and sensitivity to apoptosis. Indeed, PTEN is present in definite subcellular locations where it performs distinct functions acting on specific effectors. In this review, we summarize recent advantages in methods to study PTEN subcellular localization and the distinct biological functions of PTEN in different cellular compartments. A deeper understanding of PTEN's compartmentalized-functions will guide the rational design of novel therapies.