Cell-penetrating peptides are short cationic peptides with inherent ability to cross the plasma membrane barrier as well as intracellularly deliver cargo molecules conjugated to them. Venoms from snakes, scorpions and spiders are rich in membrane-active peptides. Crotamine from snake venom as well as maurocalcine and imperatoxin isolated from scorpion venoms have been reported to possess cell-penetrating property in mammalian cells. Latarcins, a group of spider venom toxins, has also been reported to possess antimicrobial property. However, cell-penetrating ability of Latarcins is still not elucidated. This is the first report where cell-penetrating ability of a peptide derived from spider toxin, Latarcin 1 has been demonstrated. Interestingly, the structurally minimized sequence of Latarcin 1 (LDP – Latarcin-derived peptide) when conjugated with nuclear localization sequence from Simian Virus T40 antigen (LDP-NLS) translocates across cell membrane in HeLa cells. The chimeric LDP-NLS peptide also did not exhibit cytotoxicity towards mammalian cells in contrast to the LDP that showed lesser uptake and higher cytotoxicity. LDP-NLS also successfully delivered macromolecular protein cargo inside the cells.