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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, characterized by an extremely low survival rate once diagnosed. Over 90% of cases harbor activating KRAS mutations, which are associated with metabolic reprogramming and altered endocytic activity. New treatment modalities are urgently needed. Phospholipid-shelled, gas-filled nanobubbles (NBs) are promising ultrasound (US) contrast agents and drug delivery vehicles, with potential applications in pancreatic cancer. However, their mechanisms of cellular internalization and the resulting biological responses need to be better understood before they can be effectively utilized as theranostic agents. Here, we analyzed the internalization kinetics, endocytic pathways, acoustic responses, and cell-stress-related protein expression in perfluorocarbon-filled NBs in two pancreatic cancer cell lines: KRAS-mutant PANC-1 and KRAS wild-type BxPC-3 cells showed distinct NB uptake profiles. Flow cytometry demonstrated approximately 1.5-fold higher TR-NB uptake in PANC-1 cells compared with BxPC-3 cells, suggesting enhanced cellular association and/or intracellular retention. Higher NB signal in PANC-1 cells was accompanied by weaker and less punctate LysoTracker staining, whereas BxPC-3 cells displayed brighter, more granular acidic vesicles, suggesting differences in intracellular trafficking and compartmentalization. Pharmacological inhibition of endocytosis revealed that clathrin-mediated endocytosis contributes predominantly to uptake in BxPC-3, whereas macropinocytosis appears to play a greater role in PANC-1 cells. Upon US exposure, NBs exhibited strong initial acoustic activity that declined over time. Analysis of stress-response-associated protein expression revealed cell-specific differences: PANC-1 cells exhibited higher levels of FABP-1, HSP60, SOD2, Thioredoxin-1, and SIRT2, whereas BxPC-3 cells displayed increased COX-2 and PON-2. Collectively, our findings demonstrate that NB internalization and fate differ between PDAC subtypes, potentially reflecting cell type dependent variations in endocytic, lysosomal and adaptive pathways, underscoring the importance of developing cell-tailored nanocarrier designs to enhance delivery precision and therapeutic efficacy.