Aref Teimouri,1,2 Sanaz Jafarpour Azami,1 Hossein Keshavarz,1 Fariba Esmaeili,3 Rasoul Alimi,4 Sara Ayazian Mavi,1 Saeedeh Shojaee1 1Department of Medical Parasitology and Mycology, Tehran University of Medical Sciences, Tehran, Iran; 2Students Scientific Research Center, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Medical Nanotechnology, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Background: Natural polysaccharides such as chitosan (CS) are widely used as antimicrobial agents. In recent years, and considering that CS has a strong antimicrobial potential, interest has been focused on antimicrobial activity of chitosan nanoparticles (CS NPs). The main factors affecting the antibacterial activity of chitosan include molecular weight (MW) and concentration. In this regard, the aim of this study was to produce various MWs and concentrations of CS NPs, through the ionic gelation method, and investigate their potential anti-parasitic activity against tachyzoites of Toxoplasma gondii RH strain. Materials and methods: The MWs and degree of deacetylation of the CS were characterized using viscometric and acid–base titration methods, respectively. The efficacy of various MWs and concentrations of NPs was assessed by performing in vitro experiments for tachyzoites of T. gondii RH strain, such as MTT assay, scanning electron microscopy, bioassay in mice and PCR. In vivo experiment was carried out in BALB/c mice which were inoculated with tachyzoites of T. gondii RH strain and treated with various MWs of CS NPs. Results: The results of in vitro and in vivo experiments revealed that anti-Toxoplasma activity strengthened as the CS NPs concentration increased and the MW decreased. In vitro experiment showed 100% mortality of tachyzoites at 500 and 1,000 ppm concentrations of low molecular weight (LMW) CS NPs after 180 min and at 2,000 ppm after 120 min. Furthermore, a 100% mortality of tachyzoites was observed at 1,000 and 2,000 ppm concentrations of medium molecular weight (MMW) CS NPs and at 2,000 ppm concentration of high molecular weight (HMW) CS NPs after 180 min. Growth inhibition rates of tachyzoites in peritoneal exudates of mice receiving low, medium and high MWs of CS NPs were found to be 86%, 84% and 79% respectively, compared to those of mice in sulfadiazine treatment group (positive control). Conclusion: Various MWs of CS NPs exhibited great anti-Toxoplasma efficiency against tachyzoites of RH strain, with the greatest efficacy shown by LMW CS NPs in both experiments. It seems that CS NPs can be used as an alternative natural medicine in the treatment of toxoplasmosis. Keywords: chitosan nanoparticles, molecular weights, Toxoplasma gondii RH strain, degree of deacetylation