The most widely used method for increasing uptake on macrophage is specific targeting for mannose receptor (MR) presented on macrophages. Efficiency of the uptake for MR is influenced by the space length and flexibility of mannose ligand in liposome (LP). We prepared mannosylated liposomes (M‐EGn‐LP‐ICG) encapsulated indocyanine green (ICG) with mannose ligand of various ethylene glycol units (EG), LP‐ICG, and mannosylated liposome (M‐LP‐ICG) incorporated with p‐aminophenyl‐α‐d‐mannopyranoside. We studied the effect of space length of the mannose ligand in vitro and in vivo with prepared liposomes. A space length of two ethylene glycol units at least was needed for uptake by macrophages and the uptake was increased as the space length increased up to EG4. We measured near‐infrared (NIR) fluorescence intensity by ICG and the fluorescence value of cell‐associated N‐(4‐nitrobenzo‐2‐oxa‐1,3‐diazole) (NBD) in liposome after cellular uptake. M‐EG4‐LP‐ICG showed lower NIR fluorescence intensity but higher NBD fluorescence value than M‐LP‐ICG. The result of pre‐treatment with d(+)‐mannose as an inhibitor showed significant decreasing in uptake of mannosylated LP‐ICG but no difference in LP‐ICG. These were explained that mannosylated LP‐ICG was taken up by macrophages through the MR and M‐EG4‐LP‐ICG showed more specific uptake than M‐LP‐ICG. We obtained images as time passed in the NIR range after intravenous administration using a Balb/c mouse with inflammatory model. The results showed high uptake in liver at early time and rapid degradation of mannosylated LP‐ICG. M‐EG4‐LP‐ICG was more selectively taken up by macrophages than M‐LP‐ICG. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 4545–4553, 2014.