Humic acids (HA) comprise one of the main fractions of humified organic matter, which improve the plant development by influencing, directly or indirectly some biochemical processes. We have shown that HA stimulate the lateral root development and the plasmalemma H⁺-ATPase activity, through a putative auxin-like mechanism. Here we comparatively evaluated the effects of the indol-3-acetic acid (IAA) and HA isolated from two different soils (Inseptsol and Ultisol) of ecological significance and two different organic residues (vermicompost and sewage sludge) on root development and on activities of tonoplast and plasmalemma H⁺ pumps. Root development was accessed by measuring the number of mitotic sites and lateral root emergence, the root surface area and the fresh weight. The H⁺ pumps activities were analyzed in H⁺-competent membranes vesicles isolated from roots by discontinuous sucrose gradient centrifugation. The data showed that HA (20 mg C L^-1) extracted from different sources as well as low IAA (0.1 nM) concentrations improved root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasma membrane H⁺-ATPase but also of vacuolar H⁺-ATPase and H⁺-pyrophosphatase. The vacuole is known to be required for cell expansion but has not previously implicated in hormone developmental regulation. The use of auxin inhibitors (PCIB and TIBA) suggested the HA stimulation effect on plant roots was associated with the auxin signaling pathways. To further assess the role of auxin-like activity of HA on the tonoplast H⁺ pumps activation, we analyzed low- and over-sensitive auxin tomato mutants. These results provide compelling evidences that HA affect both growth and cell differentiation trough an auxin signaling pathway. An unexplored phytohormonal action in which a concerted activation of the plasmalemma and tonoplast H⁺ pumps plays a key role in the cell expansion process triggered by IAA as well as HA, improving the root development.