We observed prominent effects of doxorubicin (Dox), an anthracycline widely used in anti-cancer therapy, on the aggregation and intracellular distribution of both partners of the H2A-H2B dimer, with marked differences between the two histones. Histone aggregation, assessed by Laser Scanning Cytometry via the retention of the aggregates in isolated nuclei, was observed in the case of H2A. The dominant effect of the anthracycline on H2B was its massive accumulation in the cytoplasm of the Jurkat leukemia cells concomitant with its disappearance from the nuclei, detected by confocal microscopy and mass spectrometry. A similar effect of the anthracycline was observed in primary human lymphoid cells, and also in monocyte-derived dendritic cells that harbor an unusually high amount of H2B in their cytoplasm even in the absence of Dox treatment. The nucleo-cytoplasmic translocation of H2B was not affected by inhibitors of major biochemical pathways or the nuclear export inhibitor leptomycin B, but it was completely diminished by PYR-41, an inhibitor with pleiotropic effects on protein degradation pathways. Dox and PYR-41 acted synergistically according to isobologram analyses of cytotoxicity. These large-scale effects were detected already at Dox concentrations that may be reached in the typical clinical settings, therefore they can contribute both to the anti-cancer mechanism and to the side-effects of this anthracycline.