Abstract Study question Are mitochondrial uncoupling proteins (UCPs) expressed in human spermatozoa and if yes, what is their role? Summary answer UCP1, UCP2, and UCP3 are expressed in human spermatozoa. The inhibition of UCPs irreversibly arrests human spermatozoa motility without compromising viability. What is known already UCPs are expressed in the mitochondrial inner membrane, where they act as channels between the intermembrane space and the matrix. Six UCP homologs had already been identified in mammals (UCP1-6). UCPs act as regulators of reactive oxygen species (ROS) production, general cellular redox state, and mitochondrial function. The altered expression or function of UCPs is positively linked with the onset of metabolic diseases, such as obesity and diabetes mellitus. Male infertility is closely related to metabolic diseases since the testis is susceptible to metabolic alterations and oxidative stress, however, the expression and function of UCPs in human spermatozoa are unknown. Study design, size, duration We performed a control-versus-treatment study. High motility spermatozoa were isolated through density gradient centrifugation from human normozoospermic seminal samples (n = 16) and incubated with genipin, a selective UCP inhibitor (0, 0.5, 5, and 50 µM), for 3 h at 37 °C. Cells and culture media were collected for analysis. Participants/materials, setting, methods UCP1-3 protein expression was detected by western blot and immunofluorescence. After UCPs inhibition, spermatozoa viability and motility were assessed. Mitochondrial membrane potential and ROS production were evaluated. Media were collected and the metabolic profile and antioxidant potential were analysed by 1H-NMR and FRAP, respectively. Main results and the role of chance We were able to identify the expression of UCP1, UCP2, and UCP3 in human spermatozoa. UCP1-3 are mainly located at the equatorial segment of the head, whereas UCP1 and UCP2 are also expressed at the spermatozoa midpiece, where mitochondria are located. The inhibition of UCPs by 50 µM genipin, resulting in the UCP3 inhibition, led to the complete and irreversible loss of motility that persisted despite washing or incubation with theophylline, a cAMP activator. These effects were associated with decreased mitochondrial membrane potential and lactate production. Interestingly, the loss of motility did not compromise spermatozoa viability. In addition, the inhibition of UCPs led to no alterations concerning ROS production, possibly due to the decreased mitochondrial activity and genipin antioxidant properties. Limitations, reasons for caution This is an in vitro study with a relatively small sample size. Genipin is considered a specific yet a general inhibitor of UCPs. The development and use of specific inhibitors for each homolog will further disclose their role in human spermatozoa motility and bioenergetics. Wider implications of the findings UCPs are expressed in human spermatozoa. UCPs are important regulators of human spermatozoa motility and metabolism. The discovery and characterization of UCPs’ role in human spermatozoa open the path for studies on ROS-related pathways and bioenergetics physiology of human spermatozoa. Trial registration number 'not applicable'