SummaryVascular plants evolved to have xylem that provides physical support for their growing body and serves as a conduit for water and nutrient transport. In a previous study, we used comparative‐transcriptome analyses to select a group of genes that were upregulated in xylem of Arabidopsis plants undergoing secondary growth. Subsequent analyses identified a plant‐specific NAC‐domain transcription factor gene (ANAC012) as a candidate for genetic regulation of xylem formation. Promoter‐GUS analyses showed that ANAC012 expression was preferentially localized in the (pro)cambium region of inflorescence stem and root. Using yeast transactivation analyses, we confirmed the function of ANAC012 as a transcriptional activator, and identified an activation domain in the C terminus. Ectopic overexpression of ANAC012 in Arabidopsis (35S::ANAC012 plants) dramatically suppressed secondary wall deposition in the xylary fiber and slightly increased cell‐wall thickness in the xylem vessels. Cellulose compositions of the cell wall were decreased in the inflorescent stems and roots of 35S::ANAC012 plants, probably resulting from defects in xylary fiber formation. Our data suggest that ANAC012 may act as a negative regulator of secondary wall thickening in xylary fibers.