AbstractMelanoma is the most aggressive and fatal type of skin cancer due to being highly proliferative. Acetylsalicylic acid (ASA; Aspirin) and salicylic acid (SA) are ancient drugs with multiple applications in medicine. Here, we showed that ASA and SA present anticancer effects against a murine model of implanted melanoma. These effects were also validated in 3D- and 2D-cultured melanoma B16F10 cells, where the drugs promoted pro-apoptotic effects. In both in vivo and in vitro models, SA and ASA triggered endoplasmic reticulum (ER) stress, which culminates with the upregulation of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). These effects are initiated by ASA/SA-triggered Akt/mTOR/AMPK-dependent activation of nitric oxide synthase 3 (eNOS), which increases nitric oxide and reactive oxygen species production inducing ER stress response. In the end, we propose that ASA and SA instigate anticancer effects by a novel mechanism, the activation of ER stress.