Little is known on the effect of application of different nitrogen (N) fertilizers on soil organic carbon (SOC) sequestration in soil humic substances (HS). We investigated HS molecular characteristics in an Orthic Acrisol, southwestern China, under 2-year field fertilization of a urea (U), a polymer-coated urea (PCU) and a biochar-coated urea (BCU) using ¹³ C-CPMAS-NMR spectroscopy. Results showed that N fertilization promoted SOC sequestration into HS and favored alkyl-C and aromatic-C rather than O-alkyl-C and carbonyl-C for humic acids and humin in soil. Application of PCU and BCU may better enhance vegetable yield, SOC sequestration, and HS stability than the U application, which may benefit from longer time of N existence and higher total N in soil. Among the N treatments, BCU application mostly affected the compositions and stability of SOC in the HS for the OC input and prime effect of biochar for SOC transformation.