2-Methoxy-1,4-naphthoquinone (MNQ) has been shown to cause cytotoxic towards various cancer cell lines. This study is designed to investigate the regulatory effect of MNQ on the key cancer genes in mitogen-activated protein kinase, phosphoinositide 3-kinase, and nuclear factor кB signaling pathways. The expression levels of the genes were compared at different time point using polymerase chain reaction arrays and Ingenuity Pathway Analysis was performed to identify gene networks that are most significant to key cancer genes. A total of 43 differentially expressed genes were identified with 21 up-regulated and 22 down-regulated genes. Up-regulated genes were involved in apoptosis, cell cycle and act as tumor suppressor while down-regulated genes were involved in anti-apoptosis, angiogenesis, cell cycle and act as transcription factor as well as proto-oncogenes. MNQ exhibited multiple regulatory effects on the cancer key genes that targeting at cell proliferation, cell differentiation, cell transformation, apoptosis, reduce inflammatory responses, inhibits angiogenesis and metastasis.