Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these “side” effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.Fil: Canals, Daniel. Stony Brook University; State University of New York;Fil: Salamone, Silvia. Stony Brook University; State University of New York;Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Nemeth, Erika. Stony Brook University; State University of New York;Fil: Aguilar, Daniel. Biomedical Research Networking Center in Hepatic and Digestive Diseases; EspañaFil: Hernandez Corbacho, María José. Stony Brook University; State University of New York;Fil: Adada, Mohamad. Stony Brook University; State University of New York;Fil: Staquicini, Daniela I.. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Arap, Wadih. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Pasqualini, Renata. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Haley, John. Stony Brook University; State University of New York;Fil: Obeid, Lina M.. Stony Brook University; State University of New York;Fil: Hannun, Yusuf A.. Stony Brook University; State University of New York