Background <p>MicroRNAs (miR) regulate post-transcriptional gene expression with an established role in cancer development, angiogenesis, invasion, and metastasis. miR-21 is an oncogenic miR that targets p53 and promotes tumor development. We aim to assess mir-21 expression in bladder cancer (BCa) and compare it to grossly normal-appearing tumor-adjacent tissue and controls.</p> Methods <p>This case–control study was implemented from December 2019 to July 2021. We collected 72 specimens of 36 BCa cases (36 specimens of tumoral tissue and 36 specimens of grossly normal-appearing adjacent tissue) and nine specimens from 9 control participants. After total mRNA extraction, microRNA expression was evaluated by Stem-loop real-time RT-PCR.</p> Results <p>Among the 45 participants, the mean ages in the BCa (36 patients) and control groups (9 patients) were 58.46 ± 16.69 and 56.89 ± 14.92, respectively. The mean expression of miR-21 was 1.8 ± 1.5, 2.8 ± 1.2, and 1.0 ± 0.5 in tumoral tissue, tumor-adjacent grossly normal tissue, and control specimens, respectively. miR-21 expression was higher in tumoral and tumor-adjacent tissues than in controls, with smokers exhibiting significantly elevated levels across all groups (<i>p</i> = 0.037). Additionally, miR-21 expression was significantly higher in high-grade versus low-grade BCa tumors (<i>p</i> = 0.025), with tumoral tissue expression levels of 1.9 ± 1.2 and 1.5 ± 1.3, respectively, and tumor-adjacent tissue levels of 2.7 ± 1.2 and 1.95 ± 1.4, respectively. In the control group, older age was associated with reduced expression of miR-21, whereas in BCa patients, the expression of miR-21 increased with aging (<i>p</i> &lt; 0.05).</p> Conclusion <p>miR-21 expression was higher in the tumor-adjacent tissue than in tumoral tissue and control specimens. The study recognizes certain limitations, such as a limited control group and possible sample loss, which might influence the strength and applicability of the findings.</p>