Proline containing peptides are highly important due to their natural abundance in various secondary structural elements like turns (β turn and γ turn etc.) in proteins. Here the conformation, cytotoxicity and structure of a unique tetrapeptide composed of alternative D- and L-proline residues are discussed. The peptide showed a polyproline II like conformation in dilute aqueous solution. The aqueous solution of the peptide self-assembled to form spheroidal oligomers with a diameter of 90 nm. The morphological features were confirmed by bright field confocal images, TEM analysis and AFM. The alternative D- and L-proline residues in the peptide showed toxicity towards cancer cell lines and 50% cell death was recorded against three different types of cancer cells (Neura 2a, HEK 293 and Hep G2).