The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the later process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and proper DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 may also be directly phosphorylated by DNA-PK at serine residue 134. While this modification does occur in undamaged cells, it is largely suppressed by PPP-family protein phosphatases. Following the disruption of replication forks, S134 phosphorylation is however enhanced and promotes cellular survival. Interestingly, while hSSB1 is known to associate with replication forks following their disruption, we here find that S134 phosphorylation may limit binding to replication fork junctions. Together, these data represent a novel mechanism for hSSB1 regulation following the inhibition of replication.