Background: Bladder cancer is a prevalent disease with significant health care costs and high rates of recurrence. Results from numerous studies to associate bladder cancer with serum biomarkers have been analyzed for prognostic indicators, or to develop agents for diagnostic and therapeutic applications. Interleukin-35 is a suppressive cytokine that has a role in tumor immunity as a regulatory cytokine by suppressing T cell anticancer responses. Methods: In the present study, we have investigated interleukin-35 serum levels in bladder cancer patients by ELISA, and compared these levels with a healthy comparison group, as well as among different clinicopathological subgroups.Results: We observed no difference in serum levels of interleukin-35 in bladder cancer patients and healthy controls; however, bladder cancer patients diagnosed at lower stages (0a, I, II) had significantly higher levels of interleukin-35 in their sera compared to high stage (III, IV) patients (P=0.018).Conclusion: Our results could indicate that interleukin-35 has no significant role in bladder cancer pathogenesis and progression. Interleukin-35 might not be a valuable biomarker for diagnosis or assessment of bladder cancer progression in clinical settings. However, further studies are needed in order to reach a definitive conclusion.