Cutaneous leishmaniasis is the most common form of leishmaniasis caused by different species of Leishmania parasites. The emergence of resistance, toxicity, long term treatment, high cost of the current drugs, and intracellular nature of the parasite are the major difficulties for the treatment of leishmaniasis. Although the therapeutic effect of paromomycin (PM) on leishmaniasisLeishmania parasite). PM-loaded into mannosylated CS (MCS) nanoparticles using dextran (PM-MCS-dex-NPs) was prepared by ionic gelation and then characterized. The particle size and Zeta potential of PM-MCS-dex-NPs were obtained as 246 nm and + 31 mV, respectively. Mannosylation of CS was qualitatively evaluated by Fourier-transform infrared spectroscopy and quantitatively measured by CHNO elemental analysis; also, a mannosylation level of 17% (w) was attained. Encapsulation efficiency (EE), drug release profile, and THP-1 cell uptake potential were determined. A value of 83.5% for EE and a higher release rate in acidic media were achieved. THP-1 cell uptake level of PM-MCS-dex-NPs after 6 h was ˜2.8 and ˜3.9 times of non-mannosylated CS nanoparticles (PM-CS-dexIn vitroGlucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs after 48 h were obtained as 1846 ± 158, 1234 ± 93, 784 ± 52 and 2714 ± 126 μg mL-1Glucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs after 48 h were obtained as 105.0 ± 14.0, 169.5 ± 9.8, 65.8 ± 7.3 and 17.8 ± 1.0 μg mL-1Glucantim, PM-CS-dex-NPs and PM-MCS-dexGlucantim, PM-CS-dex-NPs, and PM-MCS-dex-NPs at a typical concentration of 20 μg mL-1 were 71.78, 69.94, 83.14 and 33.41%, respectively. While the effect of PM-CS-dex-NPs was more salient on amastigotes, PM-MCS-dex-NPs effectively affected both stages of the parasite, especially the amastigote one. This indicated that the mannosylated formulation acts as a targeted delivery system. The findings of this study revealed that this novel targeted formulation represented a strong anti-leishmanial activity.