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Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.