p53 inactivation is a hallmark in non-small-cell lung cancer (NSCLC). It is therefore highly desirable to develop tumor-specific treatment for NSCLC therapy by restoring p53 function. Herein, a novel naphthalimide compound, NA-17, was identified as a promising drug candidate in view of both its anticancer activity and mechanism of action. NA-17 exhibited strong anticancer activity on a broad range of cancer cell lines but showed low toxicity to normal cell lines, such as HL-7702 and WI-38. Moreover, NA-17 showed p53-dependent inhibition selectivity in different NSCLC cell lines due to the activation state of endogenous p53 in the background level. Further studies revealed that NA-17 caused cell cycle arrest at the G1 phase, changed cell size, and induced apoptosis and cell death by increasing the proportion of sub-G1 cells. Molecular mechanism studies suggested that targeted accumulation of phospho-p53 in mitochondria and nuclei induced by NA-17 resulted in activation of Bak and direct binding of phospho-p53 to the target DNA sequences, thereby evoking cell apoptosis and cell cycle arrest and eventually leading to irreversible cancer cell inhibition. This work provided new insights into the molecular interactions and anticancer mechanisms of phospho-p53-dependent naphthalimide compounds.