The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell-PM, we found confined diffusion + hop movements (termed "hop diffusion") for both a non-raft phospholipid and a transmembrane (TM) protein, transferrin receptor (TfR), and the equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell-dependent), even after actin-modulating-drug treatments. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells, and demonstrated that the MSK mesh size was the same as the compartment size for the PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support the model of anchored-TM-protein pickets lining the actin-based MSK, as a major mechanism for regulating diffusion.