Previous works have reported significant effects of macromolecular crowding on the structure and behavior of biomolecules. The crowded intracellular environment, in contrast to in vitro buffer solutions, likely imparts similar effects on biomolecules. The enzyme serving as the gatekeeper for the genome, RNA polymerase (RNAP), is among the most regulated enzymes. Although it was previously demonstrated that macromolecular crowding affects association of RNAP to DNA, not much is known about how crowding acts on late initiation and promoter clearance steps, which are considered to be the rate-determining steps for many promoters. Here, we demonstrate that macromolecular crowding enhances the rate of late initiation and promoter clearance using in vitro quenching-based single-molecule kinetics assays. Moreover, the enhancement’s dependence on crowder size notably deviates from predictions by the scaled-particle theory, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions could be affected by crowded conditions in the cellular milieu.