Advances in nanoscale systems for cancer treatment have been involved in enabling highly regulated site-specific localization to sub cellular organelles hidden beneath cell membranes. So far, the cellular entry of these nanoscale systems has not been fully understood. Endocytosis is an energy-dependent process of active transport in which elected extracellular molecules (such as proteins, viruses, micro-organisms and nanoscale systems) are engulfed into the cell. This process appears at the plasma membrane surface and contains internalization of the cell membrane as well as the membrane proteins and lipids of cell. There are multiform pathways of endocytosis for nanoscale systems. Further comprehension of the mechanisms of endocytosis is achieved with a combination of efficient genetic manipulations, cell dynamic imaging and chemical endocytosis inhibitors. This review provides an account of various endocytic pathways, itemizes current methods to study endocytosis of nanoscale systems, discusses some factors associated with cellular uptake for nanoscale systems and introduces the trafficking behavior for nanoscale systems with active targeting. An insight into the endocytosis mechanism is urgent and significant for developing safe and efficient nanoscale systems for cancer diagnosis and therapy.