Ursolic acid (UA) is a naturally occurring ursane triterpenoid, which exhibits a wide range of unique biological activities. To clarify its mechanism of action (MOA), a series of fluorescent derivatives of UA (5a–c) were designed and synthesized by conjugation with 7-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore. Among them, 5cexhibited similar anti-proliferative activity with UA against HCT116 cells (half maximal inhibitory concentration (IC50) = 9.21 ± 0.50 μmol/L). Cell imaging experiment indicated that 5cwas rapidly taken up in HCT116 cells in a dose and time-dependent manner. Then, 5cwas found to localize in endoplasmic reticulum (ER), lysosomes, and mitochondria, but not in nucleus of HCT116 cells by confocal microscopy studies. Preliminary MOA proved that UA induced autophagy with a unique intracellular distribution mechanism involving ER and lysosome. In all, our work provides new clues for revealing the molecular mechanism of UA as an antitumor agent.