Abstract: Spent mushroom substrate (SMS), leftovers after cultivation of mushroom, could serve as an organic fertilizer. In this study, different proportions of SMS were mixed into paddy rice soils under laboratory conditions to study the relationship between application of SMS and soil organic carbon decomposition, and further provide reference for the rational utilization of SMS for sustainable agricultural development. The study consisted of 5 treatments-no SMS application (TS), SMS mix with paddy rice soil at 10:1 (SM1), SMS mix with paddy rice soil at 5:1 (SM2), SMS mix with paddy rice soil at 2:1 (SM3) and sole SMS medium (TM). Then changes in soil organic carbon and nitrogen, decomposition process of organic carbon in soils and CO₂ release characteristics in each treatment were determined. The results showed that soil organic carbon and total nitrogen contents under different proportions of SMS treatments were significantly higher than those under TS treatment for the same incubation time. Increase in organic carbon and total nitrogen contents mainly depended on the amount of SMS added to the soil. TM treatment showed the most obvious effect, which increased soil organic carbon and total nitrogen contents by 10.7 and 11.0 times, respectively. With increasing duration of incubation time, soil organic carbon and nitrogen decreased with the decomposition of carbon and nitrogen in all the treatments. Also organic carbon and nitrogen decreased relatively quickly under TM treatment after 35 d. The more SMS supply, the greater was the residue rate. After 63 d of cultivation, the relationships between the residue rates of organic carbon (Y_C) and nitrogen (Y_N) with the amount of SMS (X) were as follows:Y_C=71.26X-0.607 5 (r²=1.000 0^**) and Y_N=74.039X-0.413 3 (r²=0.999 9^**). The release rates of CO₂ in all the treatments increased initially and then decreased before stabilization. The higher the amount of SMS, the higher was the release rate of CO₂. On the 7^th d after cultivation, the release rate of CO₂ was highest in each treatment. After 14 d of cultivation, the release rate of CO₂ in each treatment gradually decreased at a steady state. The order of the release rate of CO₂ during the culturing period was TM > SM3 > SM2 > SM1 > TS. The cumulative release of CO₂ showed a rapid growth in the early and slowed growth in the late periods. Mineralization intensity of soil organic carbon was very small after 35 d of cultivation and most of the organic carbon was fixed in the soil. In all the treatments, TM showed the lowest organic carbon mineralization intensity, indicating that SMS was beneficial for soil carbon sequestration.