The presence of extracellular polymeric substance (EPS) plays a vital role in the accumulation and toxicity of nanoparticles to microorganisms, in which the involved processes and mechanisms are still waiting to be revealed. Herein, we specifically investigated the interfacial interaction between titanium dioxide nanoparticles (nTiO₂) and algae (Chlorella pyrenoidosa) with/without EPS and the effect of EPS on algal cell internalization of nTiO₂. Results showed that the presence of EPS on cell surface promoted heteroaggregation between nTiO₂ and algal cells, and induced more nTiO₂ accumulation on algal surface; however, algal cell internalization of nTiO₂ was limited by the presence of EPS. Pearson correlation analysis further proved that the presence of EPS had a positive effect on the surface accumulation of nTiO₂ and a negative effect on the internalization of nTiO₂. More than 60% of cell internalized nTiO₂ entered algal cells through the energy dependent endocytosis pathway. It is interesting to find that anatase nTiO₂ (nTiO₂-A) entered algal cells mainly through the clathrin dependent endocytosis, while rutile nTiO₂ (nTiO₂-R) mainly through the dynamin dependent endocytosis. This difference could be due to the different affinities of nTiO₂-A and nTiO₂-R to the mediating receptors referring to different endocytic pathways. The removal of EPS activated the associated mediating pathways, allowing more nTiO₂ to be internalized. These findings address the role of EPS on the interaction between nTiO₂ and algae and promote a deeper understanding of the ecological effect of nTiO₂.