Chemical composition of soil organic carbon (SOC) is central to many soil functions. We hypothesize that change in SOC contents resulting from various long-term fertilization strategies may accompany the shift in SOC chemical structure.  This study aims to examine the effect of fertilization strategies on the SOC composition along time of fertilizer application by ¹³C nuclear magnetic resonance (NMR) spectroscopy. The soils subjected to seven fertilizer treatments were collected in 1989, 1999, and 2009, respectively, representing 0, 10 and 20 years of fertilization. The seven fertilizer treatments were (1-3) balanced fertilization of organic compost (OM), half organic compost plus half chemical fertilizer (1/2OM), and pure chemical NPK fertilizer (NPK), (4–6) unbalanced chemical fertilization without one of the major elements (NP, PK, and NK), and (7) an unamended control (CK). The SOC contents from the balanced fertilization treatments were 2.3-52.6% and 9.4-64.6% higher than from the unbalanced fertilization/CK treatments in 1999 and 2009, respectively, indicating more significant difference with time between them. Significantly larger proportion of O-alkyl C and smaller proportion of aromatic C in the balanced fertilization than unbalanced fertilization/CK treatments was observed in 1999, but not in 2009, because their proportions from the former treatments approached the latter treatments in 2009, with no significant differences with time being found for the latter treatments. Principal components analysis further showed that the functional groups from various fertilization strategies had a trend to become compositionally similar with time of fertilizer application. The results suggest that shift in SOC chemical composition may be governed by time of fertilizer application and types of fertilization strategy.