The widespread application of titanium dioxide nanoparticles (TiO2 NPs) leads to its significant presence in sewage sludge, and its negative impact on sludge dewatering has already raised increasing concerns. However, the underlying mechanism of how TiO2 NPs deteriorate sludge dewatering is poorly understood. This work therefore aims to deeply understand how the entering TiO2 NPs affect sludge dewatering through comparing the dewatering performance among sludge withdrawn from four laboratory-scale sequencing batch reactors receiving different concentrations of TiO2 NPs. It was found that although short-term exposure to environmentally relevant level of TiO2 NPs (i.e., 1 mg/L) had no significant effect on sludge dewaterability, long-term exposure to 1 mg/L TiO2 NPs increased specific resistance to filtration (SRF) of sludge by 13.8%. When TiO2 NPs increased to 50 mg/L, short-term and long-term exposure increased SRF by 14.8% and 43.8%, respectively. The mechanism analysis revealed that the entering TiO2 NPs triggered substantial amount of reactive oxygen species, which induced cellular stress and decreased ATP generation. Cellular stress induced sludge cells to oversecrete EPS to protect them against such toxic conditions while decreases in ATP lowered active transport efficiency, which may lead to the accumulation of enzymatic products in EPS and cause shifts in microbial community such as Patescibacteria. These variations caused by TiO2 NPs had an impact on physicochemical properties of sludge, which were the main reasons for TiO2 NPs deteriorating sludge dewatering.