The effects of humic acids (HA) isolated from seven different Oxisols from Brazilian tropical and subtropical regions on root growth of maize seedlings were investigated. The HA were characterized by elemental composition, 13C solid-state nuclear magnetic resonance (NMR) (13C cross-polarization magic angle spinning-NMR), diffusion-ordered NMR spectroscopy, and high-performance size exclusion chromatography. The HA differed in hydrophobicity and molecular dimension of their alkyl, carbohydrate-like, and aromatic C components. Changes in plasma membrane H+-ATPase activity and expression in root cells were considered physiological markers of HA bioactivity. All HA promoted root growth and proton pump activation in maize vesicles. Maize plants treated for 7 days with 20 mg C HA L−1 increased adenosine triphosphate hydrolysis from 72% to 392%, and H+-ATPase expression from 111% to 495%. Within a general stimulation of root areas over control, some HA preferentially increased root length, whereas others enhanced root density. Root growth enhancement was significantly correlated to HA hydrophobicity. Although acting synergistically with hydrophobicity, the relationship to bioactivity of HA apparent molecular size and carbon content was of a lesser significance. The conformational dynamics of humic hydrophobic associations in the rhizosphere may release auxin-like plant growth promoters and actively enhance plants' biochemical activities.