Classical studies on root hydrotropism have hypothesized the importance of columella cells as well as the de novo gene expression, such as auxin-inducible gene, at the elongation zone in hydrotropism; however, there has been no confirmation that columella cells or auxin-mediated signaling in the elongation zone are necessary for hydrotropism. We examined the role of root cap and elongation zone cells in root hydrotropism using heavy-ion and laser microbeam. Heavy-ion microbeam irradiation of the elongation zone, but not that of the columella cells, significantly and temporarily suppressed the development of hydrotropic curvature. However, laser ablation confirmed that columella cells are indispensable for hydrotropism. Systemic heavy-ion broad-beam irradiation suppressed de novo expression of INDOLE ACETIC ACID 5 gene, but not MIZU-KUSSEI1 gene. Our results indicate that both the root cap and elongation zone have indispensable and functionally distinct roles in root hydrotropism, and that de novo gene expression might be required for hydrotropism in the elongation zone, but not in columella cells.