The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous (F-) actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase WRNIP1 in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized DARPins to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.