DNA inverted repeats (IRs) are hotspots of genomic instability in both prokaryotes and eukaryotes. This feature is commonly attributed to their ability to fold into hairpin- or cruciform-like DNA structures interfering with DNA replication and other genetic processes. However, direct evidence that IRs are replication stall sites in vivo is currently lacking. Here, we show by 2D electrophoretic analysis of replication intermediates that replication forks stall at IRs in bacteria, yeast, and mammalian cells. We found that DNA hairpins, rather than DNA cruciforms, are responsible for the replication stalling by comparing the effects of specifically designed imperfect IRs with varying lengths of their central spacer. Finally, we report that yeast fork-stabilizing proteins, Tof1 and Mrc1, are required to counteract repeat-mediated replication stalling. We show that the function of the Tof1 protein at DNA structure-mediated stall sites is different from its previously described effect on protein-mediated replication fork barriers.