Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Yet the precise signal transduction pathway remains poorly understood. In this study, we have investigated the molecular mechanism in NPe6-PDT-induced apoptosis in human lung adenocarcinoma cells (ASTC-a-1). A recombinant fluorescence resonance energy transfer (FRET) Bid probe was utilized to determine the kinetics of Bid cleavage. The results show that cleavage of the Bid-FRET probe occurred 150 ± 5 min after NPe6-PDT treatment, and this process lasted for 45 ± 5 min. The Bid cleavage coincided with a translocation of tBid from cytoplasm to mitochondria. Remarkably, a significant protection against cell death was observed by using small interfering RNA for Bid. Therefore, our study clearly showed the dynamics of Bid activation and redistribution during NPe6-PDT-induced apoptosis by using real-time analysis in living cells, and the inhibition of cell death by silencing Bid with interference strongly suggested that activation of Bid is required for inducing apoptosis in this experimental model.