Cisplatin and its derivatives, nitrogen mustards (NMs) and mitomycin C (MMC) are widely used in cancer chemotherapy. Their efficacy is linked primarily to their ability to generate DNA interstrand cross-links (ICLs), which effectively block the progression of transcription and replication machineries. Release of this block, referred to as unhooking, has been postulated to require endonucleases that incise one strand of the duplex on either side of the ICL. Here, we investigated how 5' flap nucleases FANCD2-associated nuclease 1 (FAN1), exonuclease 1 (EXO1) and flap-endonuclease 1 (FEN1) process a substrate reminiscent of a replication fork arrested at an ICL. We now show that EXO1 and FEN1 cleaved the substrate at the boundary between the single-stranded 5' flap and the duplex, whereas FAN1 incised it 3-4 nucleotides in the double-stranded region. This affected the outcome of processing of a substrate containing an NM-like ICL two nucleotides in the duplex region, because FAN1, unlike EXO1 and FEN1, incised the substrate predominantly beyond the ICL and thus failed to release the 5' flap. We also show that FAN1 was able to degrade a linear ICL substrate. This ability of FAN1 to traverse ICLs in DNA could help elucidate its biological function, which is currently unknown.