It is well established that the anisotropy of nanoparticles has a dominant effect on increasing their uptake by cells. However, the advantage of anisotropy for cell penetration has yet to be utilised for soft nanoparticles such as liposomes, which have wide-ranging applications in drug delivery and diagnostics. This is because their formulation is far from trivial. Here we propose a study of the morphology of nanometre-scale anisotropic liposomes we have formed by adjusting both the osmotic balance and cholesterol content, which leads to a significant proportion of rod-shaped liposomes. To our knowledge, this is the first study of the effect of cholesterol and osmotic imbalance on the morphology of nanometre-sized unilamellar liposomes. Its findings will feed directly into several projects within our group and open the door to liposomal drug delivery vectors with tuneable anisotropy.