Exosomes known as nano-sized extracellular vesicles attracted recent interests due to their potential usefulness in drug delivery. Amid remarkable advances in biomedical applications of exosomes, it is crucial to understand in vivo distribution and behavior of exosomes. Here, we developed a simple method for radiolabeling of macrophage-derived exosome-mimetic nanovesicles (ENVs) with (99m)Tc-HMPAO under physiologic conditions and monitored in vivo distribution of (99m)Tc-HMPAO-ENVs using SPECT/CT in living mice. ENVs were produced from the mouse RAW264.7 macrophage cell line and labeled with (99m)Tc-HMPAO for 1 hr incubation, followed by removal of free (99m)Tc-HMPAO. SPECT/CT images were serially acquired after intravenous injection to BALB/c mouse. When ENVs were labeled with (99m)Tc-HMPAO, the radiochemical purity of (99m)Tc-HMPAO-ENVs was higher than 90% and the expression of exosome specific protein (CD63) did not change in (99m)Tc-HMPAO-ENVs. (99m)Tc-HMPAO-ENVs showed high serum stability (90%) which was similar to that in phosphate buffered saline until 5 hr. SPECT/CT images of the mice injected with (99m)Tc-HMPAO-ENVs exhibited higher uptake in liver and no uptake in brain, whereas mice injected with (99m)Tc-HMPAO showed high brain uptake until 5 hr. Our noninvasive imaging of radiolabeled-ENVs promises better understanding of the in vivo behavior of exosomes for upcoming biomedical application.