Near-infrared (NIR) fluorescent agents hold great promise for noninvasive in vivo imaging. We have recently reported that a NIR fluorescent heptamethine dye, IR-780 iodide, exhibits unique optical properties for biomedical imaging. On the basis of this foregoing work, we further describe here the potential application of IR-780 iodide as a novel NIR agent for stem cell labeling and tracking. The labeling efficiency, subcellular localization, and the effects on cell viability and differentiation of IR-780 iodide were investigated. The in vivo distribution of stem cells after intravenous transplantation was traced by whole-body animal NIR imaging. Our results showed that IR-780 iodide exhibited superior labeling efficiency and biocompatibility with unique optical properties. Following whole-body NIR imaging, the pulmonary passage of stem cells was noninvasively visualized in rats after systemic transplantation of IR-780 iodide-labeled stem cells through intravenous delivery. With this NIR imaging method, we further confirmed that pretreatment with sodium nitroprusside (SNP), a vasodilator agent, significantly reduced the cell trapping in the lung and increased the cell passage through the lung capillaries. Our study suggests that IR-780 iodide may represent an effective NIR fluorophore for stem cell labeling and tracking.