Abstract: To investigate the impact of unidirectional freeze-thaw process on the mineralization of organic carbon in different soil layers of black soil, this study focused on a typical seasonal freeze-thaw black soil in Northeast China. By adding ¹³C-labeled glucose (initial concentration of 200 μg/g) to simulate the most common organic carbon component during spring thawing period, experiments of unidirectional freeze-thaw (from −10℃ to 10 ℃) from the outside to the inside were conducted indoors, simulating the top-down one-way freeze-thaw process in the field. The changes in CO₂ emission rate and the differences in priming effect were analyzed by comparing the gradual thawing of the outer, middle, and inner soil layers during the incubation period of 15 d at the constant temperature. The results showed that: 1) After unidirectional freeze-thaw, the soil water content in the outer layer of soil column significantly increased (about 1.20-1.27 times the initial water content), while the inner layer lost water significantly (only 78.4%-84.5% of the initial water content). The difference in the water content between the inner and outer layers was more significant in soils without glucose addtion than that with added glucose. 2) After the unidirectional freeze-thaw of soil columns without glucose addition, the average cumulative CO₂ emission in the inner layer was 138.8 μg/g, significantly higher than that in the outer layer (95.1 μg/g). However, after the unidirectional freeze-thaw of soil columns with added glucose, the CO₂ emission rate in the inner layer showed significant temporal variation, with a prominent peak in the early stage (day 4), which was 1.7 and 2.0 times of peak in the outer and middle layers, respectively. However, it rapidly declined in the later stage, thus the cumulative CO₂ emission rate was 1.2 and 1.3 times of that in the outer and middle layers, respectively. 3) After undergoing the unidirectional freeze-thaw process, the cumulative priming effect in the inner layer was only −120.60%, while that in the outer layer and the middle layer was 472.46% and 356.74%, respectively. The differences in the glucose mineralization rate and the priming effect in different soil layers after freeze-thaw of black soil indicate that the process of water migration and redistribution between frozen and thawed soil layers, as well as the differences in freeze-thaw mechanisms between layers, can change the microbial community structure and carbon source utilization strategy. This provides new insights into the carbon cycling process in black soil in non-growing seasons.