Aberrant activation of fundamental cellular processes, such as proliferation, migration and survival, underlies the development of numerous human pathophysiologies, including cancer. One of the most frequently hyperactivated pathways in cancer is the phosphoinositide 3-kinase (PI3K)/Akt signalling cascade. Three isoforms of the serine/threonine protein kinase Akt (Akt1, Akt2 and Akt3) function to regulate cell survival, growth, proliferation and metabolism. Strikingly, non-redundant and even opposing functions of Akt isoforms in the regulation of phenotypes associated with malignancy in humans have been described. However, the mechanisms by which Akt isoform-specificity is conferred are largely unknown. In the present review, we highlight recent findings that have contributed to our understanding of the complexity of Akt isoform-specific signalling and discussed potential mechanisms by which this isoform-specificity is conferred. An understanding of the mechanisms of Akt isoform-specificity has important implications for the development of isoform-specific Akt inhibitors and will be critical to finding novel targets to treat disease.