The present investigation was aimed at developing and exploring the use of mannosylated gelatin nanoparticles for the selective delivery of an anti-HIV drug, didanosine, to the target organs. The mannosylated gelatin nanoparticles (MN-G-NPs) were prepared using a two-step desolvation technique and coupled with mannose using the amino group of gelatin present on the surface of nanoparticles. The mannosylation was confirmed using infrared and nuclear magnetic resonance spectroscopy. MN-G-NPs were characterized for shape, particle size, zeta potential, and percentage drug entrapment. The size of nanoparticles was found to be in range of 248-325 nm, and maximum drug payload was found to be 40.2% to 48.5%. Average size was found to be more, but drug payload was less in the case of MN-G-NPs as compared with unconjugated nanoparticles (G-NPs). The results of the in vitro release profile demonstrated that G-NPs release a comparatively higher percentage of drug than MN-G-NPs. Cellular uptake by MN-G-NPs was 2.7 times more as compared with G-NPs. Fluorescence studies revealed the enhanced uptake of MN-G-NPs in the macrophage tissues when compared with unmodified G-NPs. Intravenous administration of free-drug solution resulted in a high concentration of drug in serum, whereas it was much less in the case of G-NPs. Coupling of the nanoparticles with mannose significantly enhanced the lung, liver, and lymph nodes uptake of drug, which is reflected in the recovery of a higher percentage of the dose from these organs following administration of MN-G-NPs in comparison to noncoupled G-NPs or free drug.