Roots respond not only to gravity but also to moisture gradient by displaying gravitropism and hydrotropism, respectively, to control their growth orientation, which helps plants obtain water and become established in the terrestrial environment. As gravitropism often interferes with hydrotropism, however, the mechanisms of how roots display hydrotropism and differentiate it from gravitropism are not understood. We previously reported MIZU-KUSSEI1 (MIZ1) as a gene required for hydrotropism but not for gravitropism, although the function of its protein was not known. Here, we found that a mutation of GNOM encoding guanine-nucleotide exchange factor for ADP-ribosylation factor-type G proteins was responsible for the ahydrotropism of Arabidopsis (Arabidopsis thaliana), miz2. Unlike other gnom alleles, miz2 showed no apparent morphological defects or reduced gravitropism. Instead, brefeldin A (BFA) treatment inhibited both hydrotropism and gravitropism in Arabidopsis roots. In addition, a BFA-resistant GNOM variant, GNM696L, showed normal hydrotropic response in the presence of BFA. Furthermore, a weak gnom allele, gnomB/E, showed defect in hydrotropic response. These results indicate that GNOM-mediated vesicular trafficking plays an essential role in hydrotropism of seedling roots.