The mechanism of action of histone deacetylase inhibitors (HDACi) is mainly attributed to the inhibition of the deacetylase catalytic activity for their histone substrates. In this study, we analyzed the abundance of class I HDACs in the cytosolic, nuclear soluble and chromatin bound cellular fractions in breast cancer cells after HDACi treatment. We found that potent N-hydroxy propenamide-based HDACi induced a concentration dependent decrease in the HDAC1 associated with chromatin and a lasting concomitant increase in cytoplasmic HDAC1 while maintaining total protein expression. No such change occurred with HDAC2 or 8, however, an increase in cytoplasmic non-phosphorylated HDAC3 was also observed. The subcellular re-equilibration of HDAC1 was subsequent to the accumulation of acetylated histones and might be cell cycle dependent. This study suggests that the biological activity of a subset of N-hydroxy propenamide-based HDACi may stem from direct competition with histone substrates of HDACs as well as from spatial separation from their substrates in the nucleus and/or change in post-translational modification status of HDACs.