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Chemical composition of soil organic carbon (SOC) is central to soil fertility. We hypothesize that change in SOC content resulting from various long-term fertilization strategies accompanies the shift in SOC chemical structure. This study examined the effect of fertilization strategies along with the time of fertilizer application on the SOC composition by 13C nuclear magnetic resonance (NMR) spectroscopy. The soils (Aquic Inceptisol) subjected to seven fertilizer treatments were collected in 1989, 1999 and 2009, representing 0, 10 and 20 years of fertilization, respectively. The seven fertilizer treatments were (1–3) balanced fertilization with application of nitrogen (N), phosphorus (P) and potassium (K) including organic compost (OM), half organic compost plus half chemical fertilizer (1/2OM), and pure chemical NPK fertilizer (NPK); (4–6) unbalanced chemical fertilization without application of one of the major elements including NP fertilizer (NP), PK fertilizer (PK), and NK fertilizer (NK); and (7) an unamended control (CK). The SOC content in the balanced fertilization treatments were 2.3–52.6% and 9.4–64.6% higher than in the unbalanced fertilization/CK treatments in 1999 and 2009, respectively, indicating significant differences in SOC content with time of fertilizer application between the two treatment groups. There was a significantly greater proportion of O-alkyl C and a lower proportion of aromatic C in the balanced fertilization than in unbalanced fertilization/CK treatments in 1999, but not in 2009, because their proportions in the former treatments approached the latter in 2009. Principal component analysis further showed that the C functional groups from various fertilization strategies tended to become compositionally similar with time. The results suggest that a shift in SOC chemical composition may be firstly dominated by fertilization strategies, followed by fertilization duration.