Xiao-Ai-Ping (XAP), a traditional oriental medicinal herb isolated from the stem of Marsdenia tenacissima (Roxb.) Wight et Arn., has been shown to induce tumor cell apoptosis. In this study, we used confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET) techniques to study the molecular mechanism of XAPinduced apoptosis in single living human lung adenocarcinoma (ASTC-a-1) cells. The efficacious apoptosis was observed at 6 h after of 100 μl XAP treatment. Further monitoring the dynamics of caspase-3 activation using FRET imaging in single living ASTC-a-1 cell expressing stably with SCAT3, a FRET probe, showed that XAP activated the caspase-3 at about 2 h after XAP treatment. These data suggest that caspase-3 activation was involved in the XAP-induced apoptosis in ASTC-a-1 cells.