The NO3- removal pathway and microorganisms change along with the height of an up-flow W-SPD bioreactor was investigated in this study. Modeling and microbial community analysis were used to analyze the denitrification behavior in W-SPD bioreactor. The results showed that NO3- removal rate matched for zero-order (R-2 > 0.97) and first order (R-2 > 0.94) combination Michaelis-Menten kinetics, whereas microbial reaction rate suited for modified logistic model (R-2 > 0.99). The excellent denitrification performance (92.5%-96.4%) and microorganisms' quantity occurred in the middle of W-SPD bioreactor. Moreover, high-throughput sequencing analysis revealed that dominant denitrifiers, carbonaceous compound degrading bacteria and fermentative bacteria co-existed in W-SPD system, which was vital for efficiently sustainable NO3- removal. Hence, aerobic degradation, heterotrophic denitrification and dissimilatory nitrate reduction to ammonium (DNRA) occurred successively along the water direction in the bioreactor, offering reasonable references for W-SPD bioreactor study and application.