Appropriate application of corn straw residues increase soil organic carbon (SOC) sequestration. However, sequestration and stabilization of added carbon (C) during corn straw transformation are not fully understood. Here, we present changes in soil humus C and humic acid (HA) molecular structure during corn straw decomposition in an incubation experiment carried out for 270 d at 25 °C. Corn straw was applied at the amount of 74.76 g per 18 kg soil (i.e., 1.57 g C·kg⁻¹), in the soil surface (CS1), incorporated within 0–10 cm (CS2), applied below 10 cm soil depth (CS3), and no corn straw applied. The results showed that after corn straw application (CS1, CS2, and CS3), the accumulation of SOC content was rapid in the first 90 d. The HA spectral results of straw-amended soils showed a slight increase in aliphatic C compounds and amino acids on day 90. On day 180, the degree of condensation was less, and aliphatic C compounds were present in large quantities in soil HA. As decomposition advanced to day 270, the aliphatic character of HA appeared to slightly weaken, and soil HA was enriched with aromatic structures. These results suggest that corn straw application enriches soil HA with more aliphatic C compounds in the early stages of decomposition, and aromatic C structures are formed in the later stage of decomposition. Incorporation of corn straw into the soil (CS2 treatment) is more conducive in increasing SOC and aliphaticity in HA during corn straw decomposition, which can potentially increase C sequestration.