Cytoplasm contains a large number of macromolecules at extremely high densities. How this striking nature of intracellular milieu called macromolecular crowding affects intracellular signaling remains uncharacterized. Here, we examined the effect of macromolecular crowding on ERK MAPK phosphorylation by MEK MAPKK. Addition of polyethylene glycol-6000 (PEG-6000) as a crowder to mimic intracellular environments, elicited a biphasic response to the overall ERK phosphorylation rate. Furthermore, probability of processive phosphorylation (processivity) of tyrosine and threonine residues within the activation loop on ERK increased non-linearly for increasing PEG-6000 concentration. Based on the experimental data, we developed for the first time a mathematical model integrating all of the effects of thermodynamic activity, viscosity, and processivity in crowded media, and found that ERK phosphorylation is transition-state-limited reaction. The mathematical model allows accurate estimation of the effects of macromolecular crowding on a wide range of reaction kinetics, from transition-state-limited to diffusion-limited reactions.