Background Transient receptor potential vanilloid 2 is a calcium channel activated by probenecid. Probenecid is a Food and Drug Administration–approved uricosuric drug that has recently been shown to induce positive lusitropic and inotropic effects in animal models through cardiomyocyte transient receptor potential vanilloid 2 activation. The aim of this study was to test the hypothesis that oral probenecid can improve cardiac function and symptomatology in patients with heart failure with reduced ejection fraction and to further elucidate its calcium‐dependent effects on myocyte contractility. Methods and Results The clinical trial recruited stable outpatients with heart failure with reduced ejection fraction randomized in a single‐center, double‐blind, crossover design. Clinical data were collected including a dyspnea assessment, physical examination, ECG, echocardiogram to assess systolic and diastolic function, a 6‐minute walk test, and laboratory studies. In vitro force generation studies were performed on cardiomyocytes isolated from murine tissue exposed to probenecid or control treatments. The clinical trial recruited 20 subjects (mean age 57 years, mean baseline fractional shortening of 13.6±1.0%). Probenecid therapy increased fractional shortening by 2.1±1.0% compared with placebo −1.7±1.0% ( P =0.007). Additionally, probenecid improved diastolic function compared with placebo by decreasing the E/E′ by −2.95±1.21 versus 1.32±1.21 in comparison to placebo ( P =0.03). In vitro probenecid increased myofilament force generation (92.36 versus 80.82 mN/mm ² , P <0.05) and calcium sensitivity (pCa 5.67 versus 5.60, P <0.01) compared with control. Conclusions Probenecid improves cardiac function with minimal effects on symptomatology and no significant adverse effects after 1 week in patients with heart failure with reduced ejection fraction and increases force development and calcium sensitivity at the cardiomyocyte level. Clinical Trial Registration URL : https://www.clinicaltrials.gov . Unique identifier: NCT 01814319.