The genome of bacteria is organized and compacted by the action of nucleoid-associated proteins. These proteins are often present in tens of thousands of copies and bind with low specificity along the genome. DNA-bound proteins thus potentially act as roadblocks to the progression of machinery that moves along the DNA. In this study, we have investigated the effect of histone-like protein from strain U93 (HU), one of the key proteins involved in shaping the bacterial nucleoid, on DNA helix stability by mechanically unzipping single dsDNA molecules. Our study demonstrates that individually bound HU proteins have no observable effect on DNA helix stability, whereas HU proteins bound side-by-side within filaments increase DNA helix stability. As the stabilizing effect is small compared to the power of DNA-based motor enzymes, our results suggest that HU alone does not provide substantial hindrance to the motor's progression in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.