Soil stores over 2500 Pg carbon (C), with the majority of C stored in deep soil layers (> 30 cm). Soil C can be lost to the atmosphere when organic compounds are mineralized to carbon dioxide (CO2, via oxidative decay or respiration) and moved upward through the soil profile (via diffusion). Soil moisture status can influence the balance between respiration and diffusion, thereby altering the soil CO2 concentration and flux. However, it is unclear how wetting and drying influence soil CO2 dynamics in surface vs. deep soil layers. Thus, we irrigated three soil profiles in Mediterranean arable land and continuously monitored soil CO2 concentration at 15, 30, 50, 70 and 90 cm during wetting and drying phases under ambient temperature conditions. We estimated gas diffusivity, CO2 flux, and temperature responses of soil CO2 concentration during the experiment. Decreases in gas diffusivity during the wetting period coincided with increases in soil CO2 concentrations. However, the negative gas diffusivity-soil CO2 concentration relationship did not hold true all the time, implying that CO2 production was the driving factor for the apparent soil CO2 concentration. We observed hysteretic responses of soil CO2 concentration to temperature as soil moisture varied, with deeper soil CO2 concentration being more sensitive to temperature than surface soil CO2 concentration, especially during the drying phase. The movement of CO2 was upward at all depths during the ambient phase, but the direction and the magnitude of CO2 fluxes varied across the depth gradient during the wetting and drying phases. This study highlights that the relative contribution of gas diffusivity vs. CO2 production to soil CO2 concentration changes with wetting and drying, and that the responses of soil CO2 concentration to temperature are dependent on the antecedent environmental conditions. Also, the downward movement of CO2 during the wetting and drying phases suggests that quantifying surface soil CO2 efflux may underestimate dynamic C processes in deeper soils.