Melanomas are one of the most aggressive form of skin cancer and are resistant to many cancer therapies. Lipid nanoparticles (LNPs) containing a pH-sensitive cationic lipid, YSK05 (YSK05-LNPs), for delivering short interfering RNA (siRNA) were found to strongly triggers in vitro toxicity in human A375 and A375-SM melanoma cell lines regardless of gene silencing. Assessing the localization of the toxicity was done by controlling the cellular uptake of the YSK05-LNPs that contained different polyethyleneglycol (PEG)-lipid. The YSK05-LNPs exhibited consistent dose- and time-dependent toxicity, independent of their cellular uptake, indicating that the toxicity is triggered by an interaction between the YSK05-LNPs and the cell surface. Treatment with free YSK05 resulted in only time-dependent toxicity. These results suggest that the YSK05-LNPs trigger two modes of action; a fast-acting component that is related to the LNP formulation and a slow-acting mode, which is related to the YSK05 lipid itself. Necrosis was determined to be the cause of cell death, as evidenced by the results of Annexin V assays, which are specific for confirming lipid-based toxicity. These findings indicate that these YSK05-LNPs have substantial potential for use as an antimelanoma agent as both an RNA interference-based drug and as a chemotherapeutic drug.