The most prevalent human carcinogen is sunlight-associated ultraviolet (UV), a physiologic dose of which generates thousands of DNA lesions per cell, mostly of two types: cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). It has not been possible, in living cells, to precisely characterize the respective contributions of these two lesion types to the signals that regulate cell cycle progression, DNA replication, and cell survival. Here we coupled multiparameter flow cytometry with lesion-specific photolyases that eliminate either CPDs or 6-4PPs and determined their respective contributions to DNA damage responses. Strikingly, only 6-4PP lesions activated the ATR-Chk1 DNA damage response pathway. Mechanistically, 6-4PPs, but not CPDs, impeded DNA replication across the genome as revealed by microfluidic-assisted replication track analysis. Furthermore, single-stranded DNA accumulated preferentially at 6-4PPs during DNA replication, indicating selective and prolonged replication blockage at 6-4PPs. These findings suggest that 6-4PPs, although eightfold fewer in number than CPDs, are the trigger for UV-induced DNA damage responses.