High mobility group (HMG) proteins of the HMGB family are small and relatively abundant chromatin-associated proteins. As architectural factors, the HMGB proteins are involved in the regulation of transcription and other DNA-dependent processes. We have examined Arabidopsis mutant plants lacking the HMGB1 protein, which is a typical representative of the plant HMGB family. In addition, our analyses included transgenic plants overexpressing HMGB1 and mutant plants that were transformed with the HMGB1 genomic region (complementation plants), as well as control plants. Both the absence and overexpression of HMGB1 caused shorter primary roots and affected the sensitivity towards the genotoxic agent methyl methanesulfonate. The overexpression of HMGB1 decreased the seed germination rate in the presence of elevated concentrations of NaCl. The complementation plants that expressed HMGB1 at wild-type levels did not show phenotypic differences compared to the control plants. Transcript profiling by microarray hybridization revealed that a remarkably large number of genes were differentially expressed (up- and down-regulated) in plants lacking HMGB1 compared to control plants. Among the down-regulated genes, the gene ontology category of stress-responsive genes was overrepresented. Neither microscopic analyses nor micrococcal nuclease digestion experiments revealed notable differences in overall chromatin structure, when comparing chromatin from HMGB1-deficient and control plants. Collectively, our results show that despite the presence of several other HMGB proteins, the lack and overexpression of HMGB1 affect certain aspects of plant growth and stress tolerance and it has a marked impact on the transcriptome, suggesting that HMGB1 has (partially) specialized functions in Arabidopsis.