Bilateral ureteral obstruction (BUO) is associated with renal damage and impaired ability to concentrate urine and is known to induce alterations in an array of kidney proteins. The aim of this study was to identify acute proteomic alterations induced by BUO. Rats were subjected to BUO for 2, 6, or 24 h. Mass spectrometry-based proteomics was performed on the renal inner medulla, and protein changes in the obstructed group were identified. Significant changes were successfully identified for 109 proteins belonging to different biological classes. Interestingly, proteins belonging to the cytoskeleton and proteins related to cytoskeletal regulation were found to be biologically enriched in BUO using online-accessible tools. Western blots confirmed the selected results, demonstrating acute downregulation of proteins belonging to all three cytoskeletal components. The microfilament protein β-actin and the intermediate filament proteins pankeratin and vimentin were all downregulated. β-Tubulin, an important microtubular protein, was found to be significantly downregulated after 24 h. Also, there was significant upregulation of cofilin, an actin-binding protein known to be upregulated in other nephropathy models. Furthermore, both upregulation and downregulation of cytoskeletal motor and regulatory proteins were observed. These findings were confirmed by immunohistochemistry, which clearly showed alterations in labeling in the inner medulla. Interestingly, we were able to confirm selected results in mpkCCD cells exposed to mechanical stretch. Our findings add to the knowledge of BUO-induced acute changes in the renal cytoskeleton and suggest that these molecular changes are partly mediated by increased stretch of the cells during obstruction.