Reports regarding the effects of long-term organic and inorganic fertilization on the quantity and quality of soil organic carbon (SOC), particularly in Vertisols, are scarce. In this study, we combined SOC physical fractionation with
13
C NMR spectroscopy technology to investigate the effect of 34 years of continuous fertilization on the SOC physical fractions and its chemical composition of 0–20 cm soil layer in a Vertisol. This study consisted of six treatments: no fertilization (control), chemical nitrogen, phosphorus and potassium fertilizers (NPK), low and high amounts of straw with chemical fertilizers (NPKLS and NPKHS), and pig or cattle manure with chemical fertilizers (NPKPM and NPKCM). Over 34 years of continuous fertilization, the SOC sequestration rate was from 0.08 Mg C ha
−1
yr
−1
in the control treatment to 0.66 Mg C ha
−1
yr
−1
in the NPKCM treatment, which was linearly related with the C input (
P
< 0.01). Of the five SOC physical fractions, two silt plus clay fractions (S + C_M, S + C_mM) dominated 74–92% of SOC, while three POM fractions (cPOM fPOM and iPOM) were only 8–26%. The two manure application treatments significantly increased all the SOC physical fractions except for the silt plus clay fraction within macroaggregates (S + C_M) compared with NPK treatment (
P
< 0.05), which was dependent on the larger amount of C input. Also, the two manure application treatments increased the levels of alkyl C and aromatic C but decreased O-alkyl C (
P
< 0.05), whereas the straw application (NPKLS and NPKHS) had no impact on the C functional groups (
P
> 0.05). Overall, the combination of animal manure with inorganic fertilization could enhance the SOC sequestration and alter its quantity and quality in Vertisols.