SummaryPlants, as predominantly sessile organisms, have evolved complex detoxification pathways to deal with a diverse range of toxic chemicals. The elasticity of this stress response system additionally enables them to tackle relatively recently produced, novel, synthetic pollutants. One such compound is the explosive 2,4,6‐trinitrotoluene (TNT). Large areas of soil and groundwater are contaminated with TNT, which is both highly toxic and recalcitrant to degradation, and persists in the environment for decades. Although TNT is phytotoxic, plants are able to tolerate low levels of the compound. To identify the genes involved in this detoxification process, we used microarray analysis and then subsequently characterized seven uridine diphosphate (UDP) glycosyltransferases (UGTs) from Arabidopsis thaliana (Arabidopsis). Six of the recombinantly expressed UGTs conjugated the TNT‐transformation products 2‐ and 4‐hydroxylaminodinitrotoulene, exhibiting individual bias for either the 2‐ or the 4‐isomer. For both 2‐ and 4‐hydroxylaminodinitrotoulene substrates, two monoglucose conjugate products, confirmed by HPLC‐MS‐MS, were observed. Further analysis indicated that these were conjugated by either an O‐ or C‐glucosidic bond. The other major compounds in TNT metabolism, aminodinitrotoluenes, were also conjugated by the UGTs, but to a lesser extent. These conjugates were also identified in extracts and media from Arabidopsis plants grown in liquid culture containing TNT. Overexpression of two of these UGTs, 743B4 and 73C1, in Arabidopsis resulted in increases in conjugate production, and enhanced root growth in 74B4 overexpression seedlings. Our results show that UGTs play an integral role in the biochemical mechanism of TNT detoxification by plants.