Translesion DNA synthesis (TLS) plays a crucial role in restarting stalled replication at damaged templates. This process is facilitated by specialized DNA polymerases, such as Polη and Polζ, where Polη inserts nucleotides opposite the damaged template, and Polζ extends the primer following insertion. TLS occurring at the stalled replication fork is termed "on-the-fly" TLS, whereas TLS that fills gaps remaining after fork progression is referred to as "post-replicative gap-filling" TLS. However, the roles of Polη and Polζ in these two phases of TLS remain unclear. Here, we demonstrate the functional relationship between Polη and Polζ in these TLS pathways through genetic studies in human cells. We established POLH−/−, REV3L−/− (deficient in Polζ catalytic subunit, Rev3), and POLH−/−/REV3L−/− cells from human TK6 cells and evaluated the sensitivity of these cell lines to ultraviolet (UV). We found that the loss of Polη in REV3L−/− cells led to the synergistic increase of the UV sensitivity, accompanied by a marked rise in UV-induced chromosomal aberrations. However, such synergistic effects were not observed in the rate of replication fork stalling after UV damage in POLH−/−/REV3L−/− cells. In marked contrast, the number of unrepaired gaps following UV irradiation was significantly increased in the double mutant. These findings suggest that Polη and Polζ function complementarily in promoting post-replicative gap-filling TLS while they act collaboratively in on-the-fly TLS. Our current genetic study in human cells revealed a previously unappreciated functional relationship between Polη and Polζ and showed the pivotal role of "post-replicative gap-filling" TLS in UV-tolerance.