The AKT kinase family is a high-profile target for cancer therapy. Despite their high degree of homology the three AKT isoforms (AKT1, AKT2 and AKT3) are non-redundant and can even have opposing functions. Small-molecule AKT inhibitors affect all three isoforms which severely limits their usefulness as research tool or therapeutic. Using AKT2-specific nanobodies we examined the function of endogenous AKT2 in breast cancer cells. Two AKT2 nanobodies (Nb8 and Nb9) modulate AKT2 and reduce MDA-MB-231 cell viability/proliferation. Nb8 binds the AKT2 hydrophobic motif and reduces IGF-1-induced phosphorylation of this site. This nanobody also affects the phosphorylation and/or expression levels of a wide range of proteins downstream of AKT, resulting in a G0/G1 cell cycle arrest, the induction of autophagy, a reduction in focal adhesion count and loss of stress fibers. While cell cycle progression is likely to be regulated by more than one isoform, our results indicate that both the effects on autophagy and the cytoskeleton are specific to AKT2. By using an isoform-specific nanobody we were able to map a part of the AKT2 pathway. Our results confirm AKT2 and the hydrophobic motif as targets for cancer therapy. Nb8 can be used as a research tool to study AKT2 signalling events and aid in the design of an AKT2-specific inhibitor.