In this study, we combined a physical fractionation procedure with natural ¹³C abundance technique to evaluate the effect of long-term fertilization on the distribution and turnover of soil organic carbon (SOC) associated with different particle size fractions. The long-term agricultural field experiment was conducted on a clay loam soil since 1959 in Southwestern Ontario, Canada. Analysis of particle size separates showed that the clay fraction contained the highest SOC content, followed by the silt fraction, and then the sand fraction. Fifty years of fertilization significantly increased the amount of SOC in all-sized fractions relative to the corresponding fractions of unfertilized soil (P<0.05). This is due to the higher maize-derived C in each fraction under fertilized soil. About 27.5-51.5% of the C was derived from maize in the fractions of fertilized soil, and only 7.9-18.0% of maize-derived C existed in the fractions of unfertilized soil. Assuming that SOC decomposed exponentially with time, we found fertilization did not significantly alter the turnover rate of C₃ plant-derived C in particle size fractions. However, C₃ plant-derived C in the sand fraction decomposed faster than that in the silt and clay fractions, especially at the surface layer. The average half-life of C₃ plant-derived C in the silt and clay fractions was 146%, 80.7%, and 44.9% longer than that in sand fraction at the 0-5, 5-10, and 10-20 cm depth, respectively. These results indicate that the old C (C₃ plant-derived) was more stable than the newly added C (C₄ plant-derived) in all size fractions; and although the sand-size C is more labile than the C in silt and clay fractions, the stability of sand-size C varies and increases greatly with soil depth.