Vallisneria-sulfur based mixotrophic denitrification (VSMD) process was put forward for the treatment of nitrate-rich agricultural runoff with low COD/TN (C/N) ratio in free water surface constructed wetland mesocosms, whose feasibility and mechanism were thoroughly studied through 273-day operation. The results showed that the average NO3--N removal efficiency and denitrification rate of VSMD mesocosms were 97.7% and 1.5gNO3--Nm-3d-1 under 5.0 or higher C/N ratio conditions in phase II (7-117d), which were similar with those of Vallisneria packed heterotrophic denitrification (VHD) mesocosms. However, VSMD mesocosms with 2.0 average C/N ratio in phase III (118-273d) were more stable and efficient than VHD mesocosms. More than 49.4mg NO3--N was reduced by VSMD mesocosms than that by VHD mesocosms throughout the operation. NO2--N accumulation in phase I (0-6d) had no influence on denitrification performance of VSMD mesocosms. In phase II and III, effluent COD, NH4+-N and NO2--N could meet the Class II standard of Environmental quality for surface water (GB3838-2002) if the experiment was carried out in batch mode. pH in VSMD mesocosms fluctuated between 7.0 and 8.9 throughout the operation without any pH buffer. The abundance of three denitrifying genes coding for nitrate (narG), nitrite (nirS), and nitrous oxide (nosZ) reductases in bottom soil and mixture from litter bags was quantified. VSMD could supply more favorable circumstances for the growth of denitrificans containing narG (3.1×108±7.9×107copiesg-1mixture-1) and nirS (2.1×108±2.0×106copiesg-1mixture-1) in litter bags than VHD, i.e., 8.7×107±1.4×107 and 1.4×108±1.5×107copiesg-1mixture-1 for narG and nirS respectively. Sulfur addition in VSMD mesocosms might increase the abundance of denitrificans containing narG and nirS, thus led to better denitrification performance.