The dataset identifies nuclear cytometry features of Feulgen-stained U-251MG glioblastoma cells cultured in the presence of sodium valproate using 5-aza-CdR treatment as a control of DNA demethylation. The cells were cultured in the presence of 10 µM lovastatin for 28 h for synchronization and then in the presence of 1- and 20-mM sodium valproate for 4 and 24 h. Treatment with 5 µM 5-aza-CdR was carried out for 28 h. The preparations were fixed in an absolute ethanol-acetic acid mixture (3:1, v/v) for 5 min and subjected to the Feulgen reaction for studying nuclear features evaluated with image cytometry using KS400-version 3 software. The features analyzed were as follows: total nuclear area (µm2), nuclear absorption area (µm2), Feret ratio, optical density (= absorbance), integrated optical density (IOD), absorbance variability per nucleus (SDtd), nuclear entropy (number of bits required to store the densitometric values per nuclear image), and nuclear energy.