Gynaecological cancer is a main subtype of cancer in women, and acquired chemoresistance is a major contributor to the poor prognosis of gynaecological cancer, but its underlying mechanism remains ill-defined. JOSD1 has been recognized as a deubiquitinase, but its biological functions remain largely unknown, especially in the context of cancer. Here we established a chemoresistant xenograft model and acquired chemoresistant cell lines to mimic the establishment of acquired chemoresistance. We identified that JOSD1 is the most upregulated DUB during the development of chemoresistance. JOSD1 depletion led to severe apoptosis in gynaecological cancer cells both in vivo and in vitro. Mechanistically, we showed that JOSD1 deubiquitinated and stabilized MCL1 to suppress mitochondrial apoptotic signalling. JOSD1 overexpression caused chemoresistance in gynaecological cancer by upregulating the MCL1 protein. Importantly, high JOSD1 expression was correlated with poor prognosis among ovarian cancer patients, and serum JOSD1 levels could be a marker for clinical diagnosis. Our study showed that JOSD1 is a novel and critical oncogene that contributes to the acquisition of chemoresistance by inhibiting mitochondrial apoptotic signalling via MCL1 stabilization. We also suggest that JOSD1 is an ideal therapeutic target and a promising diagnostic marker.