Nitrate is potentially harmful in an aquatic environment however it can be converted into other forms by microbial activities. In this research, we tested the efficiency of nitrate removal by using a denitrification bed containing two porous media; sterile soil (SS) and non-sterile soil (NSS). A Marriott column which is making constant water flux and a Plexiglas column filled with a clay loam soil, respectively. Potassium nitrate used to make different nitrate concentrations including 24, 50 and 100 (mg l− 1). The nitrate was injected up to ten pore volumes (PV( .The solute transfer parameters were estimated by using soil hydraulic parameters at HYDRUS model. As result, the growth of bacteria and the concentration of pollutants in the output of the column decreased in SS media. In the NSS with a concentration of 100 mg/ l nitrate, the number of bacteria increased to the highest, 80000 bacterial number. The presence of rival anions in the solution has a negative impact on removal of nitrate. In NSS with nitrate concentration of 24 (mg l− 1), the removal nitrate efficiency nearly 99.5 (%). Sensitivity analyses demonstrated that saturated hydraulic conductivity had the greatest effect on the variations of nitrate concentration. Besides, the correlation coefficient between simulated and observed values varied between 0.90 and 0.99. Overall, the NSS with an inlet nitrate concentration of 24 (mg l− 1), the highest nitrate removal efficiency was obtained. The total amount of bacteria of NSS at the end of the experiment has been increased because of the bacteria growth.