Homologous recombination (HR) is a major mechanism to repair DNA double-strand breaks (DSBs).Although tumor suppressor CtIP is critical for DSB end resection,a key initial event of HR repair,the mechanism regulating the recruitment of CtIP to DSB sites remains largely unknown.Here,we show that acidic nucleoplasmic DNA-binding protein 1 (And-1) forms complexes with CtIP as well as other repair proteins,and is essential for HR repair by regulating DSB end resection.Furthermore,And-1 is recruited to DNA DSB sites in a manner dependent on MDC1,BRCA1 and ATM,down-regulation of And-1 impairs end resection by reducing the recruitment of CtIP to damage sites,and considerably reduces Chkl activation and other damage response during HR repair.These findings collectively demonstrate a hitherto unknown role of MDC1→And-1→CtIP axis that regulates CtIP-mediated DNA end resection and cellular response to DSBs.