Ultrasonically-stimulated microbubbles enhance the therapeutic effects of various chemotherapy drugs. However, the application of ultrasound and microbubbles (USMB) for enhancing the therapeutic effect of nucleoside analogs, which are used as front-line treatments in a range of cancers, and its underlying mechanism is not well understood. This study investigated the effect of gemcitabine, a nucleoside analog drug, in combination with USMB in increasing cell cytotoxicity relative to either treatment alone in BxPC3 pancreatic cancer cells. Cells were sonicated using low frequency (0.5MHz) ultrasound in combination with Definity® microbubbles (1.7% v/v) in the presence of 1μM of gemcitabine for a total of 2h. USMB in combination with gemcitabine decreased cell viability (48h) to 44.7±5.2%, 27.7±4.3%, and 12.5±3.4% with increasing ultrasound peak negative pressures (220, 360, 530kPa) from 84.7±3.6%, 54.2±3.8%, and 26.8±3.0%, respectively, when USMB was applied in the absence of drug. We further confirmed that USMB did not enhance the internalization of 1μM of a radiolabeled nucleoside analog (2-chloroadenosine) at each of the three chosen ultrasound PNPs, determined by radiolabeled scintillation counting. These data suggest that USMB in combination with nucleoside analog drugs leads to an additive effect on cell toxicity and that USMB does not impair transporter-mediated uptake of nucleoside analogs.