Self-assembly of synthetic molecules has been drawing broad attention as a novel emerging approach in drug discovery. Here, we report selective cell death induced by a novel peptide amphiphile that self-assembles to form entangled nanofibers (hydrogel) based on intracellular pH (pH(i)). We found that a palmitoylated hexapeptide (C-16-VVAEEE) formed a hydrogel below pH 7. The formation of the nanofibrous self-assembly was responsive to a small pH change around pH 7. The cytotoxicity of C-16-VVAEEE was correlated with pH(i) of cells. Microscope observation demonstrated the self-assembly of C(16 )VVAEEE inside HEK293 cells. In vivo experiments revealed that the transcutaneous administration of C-16-VVAEEE showed remarkable anti-tumor activity. This study proposes that distinct microenvironment inside living cells can be used as a trigger for the intracellular self-assembly of a peptide amphiphile, which provide a new clue to drug discovery.