Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P bindsto G-protein-coupled receptors (S1PR1–5) to regulate cellular events, including Ca2+signaling. The SK1/S1P axisand Ca2+signaling both play important roles in health and disease. In this respect, Ca2+microdomains at themitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis.Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with ma-lignancies. We show that overexpression of SK1 augments agonist-induced Ca2+release from the ER resulting inincreased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely throughincreased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments in-creases mitochondrial matrix Ca2+during agonist stimulation, mimicking the SK1 overexpression in cells.Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2fragmentation resulting in increased mitochondrial Ca2+and downstream cellular effects.