Bladder cells are constantly exposed to multiple xenobiotics and bioactive metabolites. In addition to this challenging chemical environment, they are also exposed to shear stress originating from urine and interstitial fluids. Hence, physiological function of bladder cells relies on a high biochemical and biomechanical adaptive competence, which, in turn, is largely supported via autophagy-related mechanisms. As a negative side of this plasticity, bladder cancer cells are known to adapt readily to chemotherapeutic programs. At the molecular level, autophagy was described to support resistance against pharmacological treatments and to contribute to the maintenance of cell structure and metabolic competence. In this study, we enhanced autophagy with rapamycin (1–100 nM) and assessed its effects on the motility of bladder cells, as well as the capability to respond to shear stress. We observed that rapamycin reduced cell migration and the mechanical-induced translocation potential of Krüppel-like transcription factor 2 (KLF2). These effects were accompanied by a rearrangement of cytoskeletal elements and mitochondrial loss. In parallel, intracellular acetylation levels were decreased. Mechanistically, inhibition of the NAD + -dependent deacetylase sirtuin-1 (SIRT1) with nicotinamide (NAM; 0.1–5 mM) restored acetylation levels hampered by rapamycin and cell motility. Taken together, we described the effects of rapamycin on cytoskeletal elements crucial for mechanotransduction and the dependency of these changes on the mitochondrial turnover caused by autophagy activation. Additionally, we could show that targeted metabolic intervention could revert the outcome of autophagy activation, reinforcing the idea that bladder cells can easily adapt to multiple xenobiotics and circumvent in this way the effects of single chemicals.