IR-783, a near-infrared heptamethine cyanine dye, has been reported to possess cancer targeting and anticancer effects; However, the molecular mechanism by which IR-783 exhibits anti-breast cancer activity is unclear. In the present study, the inhibitory effects of IR-783 on the proliferation and migration of breast cancer cells were investigated. Our results revealed that IR-783 inhibited MDA-MB-231 and MCF-7 cell proliferation in a dose- and time-dependent manner by inducing cell cycle arrest at the G0/G1 phase. In addition, a Transwell assay demonstrated that IR-783 treatment suppressed the migratory ability of MDA-MB-231 and MCF-7 cells. Furthermore, IR-783 treatment decreased the expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in MDA-MB-231 cells. Furthermore, IR-783 induced MDA-MB-231 and MCF-7 cell mitochondrial fission, and also decreased the levels of ATP. This was accompanied with a decrease in polymerized filamentous actin, which is the fundamental component of filopodia at the cell surface. Collectively, the results of the present study demonstrated that IR-783 inhibited the proliferation and migration of MDA-MB-231 and MCF-7 cells by inducing mitochondrial fission and subsequently decreasing ATP levels, resulting in cell cycle arrest and filopodia formation suppression. These findings suggest that IR-783 may be developed into an effective novel drug for treating breast cancer.