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Long-term agricultural research with annual and perennial cropping systems provides significant insight into the carbon sequestration potential of agriculture in the East Mediterranean region of Anatolia. This study explored the effect of different application rates of phosphorus (P) fertilizer on the storage and quality of soil organic carbon (SOC) and soil CO2 flux to assess the relationship between SOC and P application. The study was based on the hypothesis that long-term P fertilization can affect carbon sequestration in soil and impact soil CO2 flux. The objectives of the present study were to: (i) analyze the effect of long-term inorganic chemical P fertilization on SOC content and (ii) estimate the effect of long-term P fertilization on CO2 flux. Under crop rotation, corn (Zea mays L.) and wheat (Triticum aestivum L.) (CW system) were exposed to four fertilizer doses (0, 50, 100, and 200 kg ha−1), which were repeated annually over 20 years. During plant growth, bare soil and treated soil CO2 fluxes were determined several times. The results demonstrated that P fertilizer did not have a direct impact on the SOC of the summer-maize growing season and had a positive impact on SOC during the winter-wheat growing season. P fertilization levels have positive impact on atmospheric CO2 capture and increased SOC content. [ABSTRACT FROM AUTHOR]