The yeast heterodimeric Mus81Mms4 complex possesses a structure-specific endonuclease activity that is critical for the restart of stalled replication forks and removal of toxic recombination intermediates. Previously, we reported that Mus81Mms4 and Rad27 (yeast FEN1, another structure-specific endonuclease) showed mutual stimulation of nuclease activity. In this study, we investigated the interactions between human FEN1 and MUS81EME1 or MUS81EME2, the human homologs of the yeast Mus81Mms4 complex. We found that both MUS81EME1 and MUS81EME2 increased the activity of FEN1, but FEN1 did not stimulate the activity of MUS81EME1/EME2. The MUS81 subunit alone and its N-terminal half were able to bind to FEN1 and stimulate its endonuclease activity. A truncated FEN1 fragment lacking the C-terminal region that retained catalytic activity was not stimulated by MUS81. MichaelisMenten kinetic analysis revealed that MUS81 increased the interaction between FEN1 and its substrates, resulting in increased turnover. We also showed that, after DNA damage in human cells, FEN1 co-localizes with MUS81. These findings indicate that the human proteins and yeast homologs act similarly, except that the human FEN1 does not stimulate the nuclease activities of MUS81EME1 or MUS81EME2. Thus, the mammalian MUS81 complexes and FEN1 collaborate to remove the various flap structures that arise during many DNA transactions, including Okazaki fragment processing.