Cells treated with ultraviolet (UV) radiation undergo cell cycle arrest at the S-phase and G1/S boundary, allowing DNA repair to occur. Several proteins such as replication protein A and DNA-dependent protein kinase have been suggested to be involved in UV-induced inhibition of DNA replication. However, the role of these proteins in inhibiting DNA replication remains unknown. Other proteins may play important roles in modulating functions of these proteins in response to UV-irradiation. To understand the broad range of proteins involved in this inhibition, we carried out a systematic study to identify specific proteins involved in UV-induced replication arrest using two-dimensional gel electrophoresis and mass spectrometry. Unique changes in protein expression level for 31 proteins were observed over a 24-hour time course, including calgizzarin, cyclophilin A, and macrophage migration inhibitory factor. The expression level changes of these proteins are dynamically correlated to DNA replication activity, suggesting involvement of these proteins in modulating DNA replication and repair activities. This proteomic approach provides opportunities to gain insights into the mechanism by which DNA replication is inhibited.