To enhance the nitrate removal in constructed wetlands (CWs) for treating hydroponic wastewater dis-charged from greenhouses, the effectiveness of HF (horizontal flow)-HF hybrid CWs utilizing a combinedsulfur-based autotrophic (based on the optimum conditions from batch experiment) and heterotrophicdenitrification was evaluated for treating hydroponic wastewater containing high nitrate and low organiccarbon concentrations. The optimum ratio of sulfur: limestone:immobilized bead with Thiobacillus deni-trificans (T. denitrificans) was found to be 3:1:4; the optimum initial cell density was above 1 × 106cells;the optimum temperature was 25–35◦C; and the optimum sulfur sources were thiosulfate and ele-mental sulfur to effectively treat hydroponic wastewater utilizing autotrophic denitrification with T.denitrificans in batch experiments. In the HF–HF CWs utilizing the combined autotrophic and het-erotrophic denitrification, the average removal efficiencies of nitrate were higher in the order of T2(71.5%, thiosulfate treatment—combination of heterotrophic and autotrophic denitrification) >T3 (66.6%,element sulfur treatment—combination of heterotrophic and autotrophic denitrification) T1 (43.0%,control—heterotrophic denitrification only). In the HF–HF CWs, the maximum nitrate removal efficiencyby the thiosulfate treatment was slightly greater than that by the treatment with elemental sulfur,whereas the sulfate production influence on autotrophic denitrification by elemental sulfur (SO42−:89.1 mg L−1) was lower as compared to thiosulfate (SO42−: 38.3 mg L−1). Because the sulfate productionis an important factor to meet acceptable drinking water quality discharge standard (Sulfate concentra-tion in the effluent was below 250 in US EPA, and 200 mg L−1in South Korea), elemental sulfur was amore suitable sulfur source in HF–HF hybrid CWs. Overall, a combined process of using E/L/B (elementsulfur/limestone/immobilized bead with T. denitrificans) column in HF–HF hybrid CWs would promoteautotrophic and heterotrophic denitrification. Therefore, a combined autotrophic and heterotrophicdenitrification process in HF–HF CWs would be more suitable than the heterotrophic denitrificationalone (conventional technology in CWs) for treating nitrate in hydroponic wastewater since hydroponicwastewater contains little organic carbon.