Anammox process has brought about cost-effective, eco-friendly, and innovative technologies to wastewater treatment by reducing the operational cost of treatment plants and decreasing greenhouse gas emissions. Titanium dioxide (TiO2), as one of the most prevalent nanoparticles (NPs) in the world, is being used in various consumer products and applications. In recent years, studies have focused on potential toxicological impacts of NPs on biological processes due to their endless production and consumption. In this context, the first time in the literature, 24 h acute TiO2 NPs exposure on Anammox process was investigated. Deterioration on anammox activity gradually increased with increasing applied TiO2 NPs concentration. At 300 mg/L exposure dose, nitrogen removal rate dramatically decreased to 37.09 ± 0.24 mgN/ gVSS.d and a severe inhibition (80.57% ± 1.17%) was observed. Among the several curve fit models, non-linear second order polynomial (quadratic) model was the best fit one with IC50 of 154 mg/L. Scanning electron microscope (SEM) images demonstrated the tendency of TiO2 NPs to aggregate and attach to the surface of the bacteria. Extracellular polymeric substance (EPS) response of anammox bacteria was also investigated and it was found that, the total EPS content gradually decreased by increasing TiO2 NPs concentration.