Intracellular protein-protein interactions provide a major therapeutic target for the development of peptide-based anticancer therapeutic agents. MDM2 is the 491-residue protein encoded by the MDM2 oncogene. Being a ubiquitin-protein ligase, MDM2 represses the transcription ability of the tumor suppressor p53 by proteasome-mediated degradation. Under typical cellular circumstances, a sustained p53 expression level is maintained by negative regulation of MDM2, whereas under stress conditions, this is alleviated to increase the p53 level. Modulation of MDM2-p53 interaction via fabrication of an MDM2-interacting peptide could be a useful strategy to inhibit subsequent proteasomal degradation of p53 and initiation of p53 signaling leading to the initiation of p53-mediated apoptosis of tumor cells. Here, in this research work, a novel anticancer peptide mPNC-NLS targeting the nucleus and the MDM2 protein (p53 negative regulator) was designed to promote the p53 protein activity for the prevention of cancer. It induces effective apoptosis in both A549 and U87 cells and remains non-cytotoxic to normal lung fibroblast cells (WI38). Further, immunocytochemistry and Western blot results confirm that the designed mPNC-NLS peptide induces the apoptotic death of lung cancer cells via activation of p53 and p21 proteins and remarkably stifled the in vitro growth of 3D multicellular spheroids composed of A549 cells.