SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites. Display omitted •SLFN11 binds replication forks in response to replication stress•SLFN11 blocks replication regardless of ATR-CHK1 activity•SLFN11 opens chromatin in the near vicinity of replication initiation sites•By killing cells with defective replication, SLFN11 arises a guardian of the genome SLFN11 is a dominant determinant of sensitivity to DNA-targeted therapies. Murai et al. show that SLFN11 is recruited to stressed replication forks, opens chromatin, and blocks replication when replication is perturbed by DNA damage or improperly activated by cell cycle checkpoint inhibition. SLFN11 emerges as a unique S-phase regulator.