BACKGROUND: We aimed to investigate the cytotoxic and apoptotic effects of miltefosine on RH strain by various techniques. METHODS: The study was conducted at the Department of Parasitology and Mycology, Urmia University of Medical Sciences, Iran in 2020. Four groups of five BALB/c mice were selected. The cytotoxicity test was conducted by adding miltefosine to tachyzoites; control tachyzoites received PBS and MTT assay was done on each suspension. For evaluating the Th1-type immune responses, the serum levels of IFN-γ and nitric oxide (NO) were assessed in mice after injecting tachyzoites and miltefosine, respectively. The flow cytometry technique was performed on tachyzoites challenged with IC and IC doses of miltefosine and unchallenged cells. DNA fragments in tachyzoites were detected by Terminal dUTPnick-end labeling (TUNEL) method. RESULTS: Overall, 256, 64, 32, and 16 μg concentrations of miltefosine, respectively could kill more than 50% of viable tachyzoites. The infected mice group, treated with miltefosine, significantly produced more IFN-γ relative to other groups (< 0.001). Moreover, a significant difference was found in inducible NO synthase between the experimental and control groups (<0.05). The flow cytometry results demonstrated a concentration-dependent apoptosis rate in tachyzoites incubated with miltefosine, though the necrosis rate was non-significant. DNA fragmentation analysis indicated oligonucleotides (18-200 bp) in tachyzoites treated with 11μg of miltefosine for 24, 48 and 72 h. However, this pattern was not observed in untreated control microorganisms. CONCLUSION: Miltefosine could be a favorable candidate for use as a new treatment for toxoplasmosis.