Root hairs are reported to be plastic in response to nutrient supply, but relatively little is known about their development in response to magnesium (Mg) availability. Here, we showed that development of root hairs of Arabidopsis decreased progressively with increasing Mg supply, which was related to the initiation of new trichoblast files and likelihood of trichoblasts to form hairs. Tip-focused reactive oxygen species (ROS) and cytosolic Ca(2+) concentrations [(Ca(2+) )c] during elongation of root hairs were enhanced under low Mg but decreased under high Mg. Under low Mg, application of diphenylene iodonium (DPI) or BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] blocked the enhanced development of root hairs and the opposite was true when the plants under high Mg were treated with phenazine methosulphate (PMS), methyl viologen (MV) or CaCl2 . Furthermore, Mg availability did not alter root hair growth in rhd2-1 mutant that contains lower levels of ROS and cytosolic [Ca(2+) ]c. Transcriptome data and qPCR results revealed a greater fraction of morphogenetic H-genes, and cell wall organization genes were up-regulated by low Mg but down-regulated by high Mg. Our data suggest a profound effect of Mg supply on the development of root hairs in Arabidopsis, through the characterized Ca(2+) and ROS signals that modulate the elongation of root hairs and the expression of root-hair morphogenetic genes.