The effect of soil organic matter (SOM) on aggregation of variably-charged red soils (Ultisol) through clay zeta potential is not fully understood. Therefore, the objectives of this study were to investigate the SOM effect on the clay zeta potential and soil aggregation after fertilization. Soils under 17 years of fertilization (manure, NPK + straw, NPK, and control (CK) were adjusted by KCl solution to reach varying soil pH and concentration in order to determine clay zeta potential, cations, and aggregate size distribution. The SOM content and C-functional groups by¹³C-NMR analysis were also determined. Results showed that the negative zeta potential displayed a bell-shaped pattern with increasing concentration of KCl, but displayed different amplitude of variation among treatments. Manure had the highest zeta potential value and its degree of variation in relative to the value at KCl concentration of 0.1 mol L⁻¹(19%), NPK + straw and NPK treatments were similar, and CK was the least. Greater negative zeta potential for manure treatment was attributed to higher SOM content, aromatic-C functional groups, and their greater concentrations of Ca²⁺and Mg²⁺than did the CK. As a result, higher SOM and clay zeta potential yielded in less release of amount of soil particles (< 10 μm) (r = − 0.46*) and enhanced water stable macroaggregates for manure instead of NPK + straw. Long-term manure fertilization would be suggested as a conservation practice for red soil due to its increase in soil aggregate stability and negative zeta potential in subtropical climate.