The role of endothelin receptor type B (EDNRB) isoform 3 involved in Temozolomide (TMZ)-induced melanoma cell death has not yet been elucidated. The subcellular localization of EDNRB isoform 3 was determined by confocal and immunoblotting assays. Silencing EDNRB isoform 3 was performed by CRISPR/Cas9. Apoptosis was assessed by annexin V/propium iodide staining and caspases 3/7/9 activity. Mitochondrial membrane potential, reactive oxygen species and mitochondrial Ca2+ were measured by flow cytometry. Apoptosis protein array was applied. Confocal and immunoblot analyses indicate mitochondrial localization of EDNRB isoform 3 and the first N-terminal (1-22) amino acids are sufficient for its mitochondrial targeting. EDNRB isoform 3 depleted A375 cells significantly confers chemoresistance with mitochondrial depolarization, reduced reactive oxygen species, enhanced mitochondrial Ca2+ uptake and decreased caspase 9 activation. Additionally, apoptosis array shows that lack of EDNRB isoform 3 has relatively lower expression of phosphorylation of p53 at S392 and a slightly higher expression of Paraoxonase 2. Our findings raise the possibility of targeting EDNRB isoform 3 as a new therapeutic strategy in combination with TMZ for melanoma treatment.