Cellular uptake is limiting for the efficacy of many cytotoxic drugs used to treat cancer. Identifying endocytic mechanisms that can be modulated with targeted, clinically-relevant interventions is important to enhance the efficacy of various cancer drugs. We identify that flotillin-dependent endocytosis can be targeted and upregulated by ultrasound and microbubble (USMB) treatments to enhance uptake and efficacy of cancer drugs such as cisplatin. USMB involves targeted ultrasound following administration of encapsulated microbubbles, used clinically for enhanced ultrasound image contrast. USMB treatments robustly enhanced internalization of the molecular scaffold protein flotillin, as well as flotillin-dependent fluid-phase internalization, a phenomenon dependent on the protein palmitoyltransferase DHHC5 and the Src-family kinase Fyn. USMB treatment enhanced DNA damage and cell killing elicited by the cytotoxic agent cisplatin in a flotillin-dependent manner. Thus, flotillin-dependent endocytosis can be modulated by clinically-relevant USMB treatments to enhance drug uptake and efficacy, revealing an important new strategy for targeted drug delivery for cancer treatment.