DNA replication stress-mediated activation of the ATR kinase pathway is important for maintaining genomic stability. In this study, we identified a zinc finger protein, ZFP161 that functions as a replication stress response factor in ATR activation. Mechanistically, ZFP161 acts as a scaffolding protein to facilitate the interaction between RPA and ATR/ATRIP. ZFP161 binds to RPA and ATR/ATRIP through distinct regions and stabilizes the RPA–ATR–ATRIP complex at stalled replication forks. This function of ZFP161 is important to the ATR signaling cascade and genome stability maintenance. In addition, ZFP161 knockout mice showed a defect in ATR activation and genomic instability. Furthermore, low expression of ZFP161 is associated with higher cancer risk and chromosomal instability. Overall, these findings suggest that ZFP161 coordinates ATR/Chk1 pathway activation and helps maintain genomic stability.