Polo-like kinase 1 (PLK1), a critical cell cycle regulator, has been identified as a potential target in osteosarcoma (OS). 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), a prostaglandin derivative, has shown its anti-tumor activity by inducing apoptosis through reactive oxygen species (ROS)-mediated inactivation of v-akt, a murine thymoma viral oncogene homolog, (AKT) in cancer cells. In the study analyzing its effects on arthritis, 15d-PGJ2 mediated shear-induced chondrocyte apoptosis via protein kinase A (PKA)-dependent regulation of PLK1. In this study, the cytotoxic effect and mechanism underlying 15d-PGJ2 effects against OS were explored using OS cell lines. 15d-PGJ2 induced significant G2/M arrest, and exerted time- and dose-dependent cytotoxic effects against all OS cell lines. Western blot analysis showed that both AKT and PKA-PLK1 were down-regulated in OS cell lines after treatment with 15d-PGJ2. In addition, transfection of constitutively active AKT or PLK1 partially rescued cells from 15d-PGJ2-induced apoptosis, suggesting crucial roles for both pathways in the anti-cancer effects of 15d-PGJ2. Moreover, ROS generation was found treatment with 15d-PGJ2, and its cytotoxic effect could be reversed with N-acetyl-l-cysteine. Furthermore, inhibition of JNK partially rescued 15d-PGJ2 cytotoxicity. Thus, ROS-mediated JNK activation may contribute to apoptosis through down-regulation of the p-Akt and PKA-PLK1 pathways. 15d-PGJ2 is a potential therapeutic agent for OS, exerting cytotoxicity mediated through both AKT and PKA-PLK1 inhibition, and these results form the basis for further analysis of its role in animal studies and clinical applications.