Triple-negative breast cancer (TNBC) lacks effective targeted therapies due to its aggressive nature. We identified DH20931, a novel small-molecule agonist of ceramide synthase 2 (CerS2) that synthesizes pro-apoptotic very long-chain ceramides (VLCCs). DH20931 directly activates CerS2 with nanomolar potency, causing VLCC accumulation and lipotoxic endoplasmic reticulum (ER) stress that triggers apoptosis via ATF4/CHOP/PUMA signaling. Mechanistically, DH20931 enhances CerS2 interaction with IP3R1 calcium channels, promoting ER-mitochondria proximity and massive calcium flux into mitochondria. This mitochondrial calcium overload induces apoptosis. In preclinical studies, DH20931 potently inhibited TNBC cell growth in 2D/3D cultures and suppressed tumor progression in orthotopic and patient-derived xenograft models with favorable safety profiles. These findings validate CerS2 as a druggable target and establish a therapeutic strategy combining ER stress with calcium-mediated mitochondrial dysfunction.