DNA replication checkpoint (Mec1-Mrc1-Rad53 in budding yeast) is an evolutionarily conserved surveillance system to ensure proper DNA replication and genome stability in all eukaryotes. Compared to its well-known function as a mediator of replication checkpoint, the exact role of Mrc1 as a component of normal replication forks remains relatively unclear. In this study, we provide in vitro biochemical evidence to support that yeast Mrc1 is able to enhance the activity of DNA polymerase ϵ (Pol ϵ), the major leading strand replicase. Mrc1 can selectively bind avidly to primer/template DNA bearing a single-stranded region, but not to double-stranded DNA (dsDNA). Mutations of the lysine residues within basic patch 1 (BP1) compromise both DNA binding and polymerase stimulatory activities. Interestingly, Mrc1-3D, a mutant mimicking phosphorylation by the Hog1/MAPK kinase during the osmotic stress response, retains DNA binding but not polymerase stimulation. The stimulatory effect is also abrogated in Mrc1 purified from cells treated with hydroxyurea (HU), which elicits replication checkpoint activation. Taken together with previous findings, these results imply that under unperturbed condition, Mrc1 has a DNA synthesis stimulatory activity, which can be eliminated via Mrc1 phosphorylation in response to replication and/or osmotic stresses