Abstract INTRODUCTION: Diabetes mellitus is the most common metabolic and endocrine disease. Depressed Heart Rate Variability (HRV) is an early warning sign of diabetic neuropathy. In numerous studies, spironolactone improved HRV and decreased mortality in Congestive Heart Failure (CHF). This study was performed to assess the effect of spironolactone on HRV in diabetic patients. methods: This prospective, randomized, double-blind clinical trial was performed on 62 diabetic patients with autonomic neuropathy at Isfahan Cardiovascular Research Center. Baseline HRV was measured with time domain and frequency domain methods using a Valiance system manufactured by US Biomedical Systems Inc. (2000). The patients were then randomly placed in case and control groups. The control group was given placebo and the case group was given 25mg spironolactone twice daily for two months. HRV was measured at the end of this period and data were analyzed using SPSS. HRV before and after medication was compared with t-test, paired t-test, Wilcoxon test, and Mann-Whitney test. results: Twenty-nine patients in the control group and thirty-three patients in the case group were assessed. HRV was measured before and after the study. T-test and Mann-Whitney test revealed no significant difference between HRV in the two groups. Paired t-test and Wilcoxon test did not show any significant difference of HRV within the two groups. Discussion: In this study, spironolactone did not improve HRV in diabetic patients.