Fragile X syndrome, a common form of inherited mental retardation, is caused by loss of the fragile X mental retardation protein (FMRP). As a selective RNA-binding protein, FMRP is localized predominately in cytoplasm, where it regulates translational control. However, there is a small portion of FMRP present in the nucleus, and its function there has been elusive. Here we show that Drosophila dFMR1 in nucleus is required for replication stress-induced H2Av phosphorylation in the DNA damage response (DDR). Replication stress could induce the expression of dFmr1 and promote the nuclear accumulation of dFMR1. We show that, upon the stimulation of replication stress, dFMR1 is associated with chromatin in a domain-specific manner, which is essential for its ability to induce the phosphorylation of H2Av. These results together reveal an unexpected nuclear role of FMRP in DDR and uncover a feed-forward mechanism by which dFmr1 and early DDR induced by replication stress reciprocally regulate each other, thereby synergistically triggering activity of the DDR signaling cascade.