The anti-apoptotic ability of Mcl-1 Delta 127, a caspase cleavage product of Mcl-1, is debated. We here used fluorescence imaging to assess the anti-apoptotic capacity of Mcl-1 Delta 127 in living cells. Fluorescence imaging of living cells expressing CFP-Mcl-1 Delta 27 showed that Mcl-1 Delta 27 existed mainly in cytoplasm. Fluorescence imaging of living cells co-expressing CFP-Mcl-1 Delta 127 and YFP-Bak, CFP-Mcl-1 Delta 127 and YFP-BimL, CFP-Mcl-1 Delta 27 and YFP-Puma or CFP-Mcl-1 Delta 27 and YFP-tBid showed that Mcl-1 Delta 27 markedly inhibited the oligomerization of Bak, BimL, Puma and tBid on mitochondria and also inhibited the Bak-, BimL-, Puma- or tBid-mediated cell death, resulting in their partial localization in cytoplasm. Fluorescence resonance energy transfer (FRET) imaging proved that Mcl-1 Delta 127 bound to Bak, BimL, Puma and tBid, respectively. Fluorescence loss in photobleaching (FLIP) analyses showed that Mcl-1 Delta 127 did prevent Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Collectively, Mcl-1 Delta 27 has the same anti-apoptotic capacity as Mcl-1, and prevents apoptosis by sequestering BH3-only or Bak proteins, thus inhibiting their oligomerization on mitochondria. (C) 2020 Elsevier Inc. All rights reserved.