Deficiencies in tricarboxylic acid (TCA) cycle enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. In mammalian spermatozoa mitochondria, the TCA cycle is known to be a crucial metabolic pathway that contributes to produce ATP. There is little known about the role and mechanism of mitochondrial aconitase (ACO2), which is an important regulatory enzyme of the TCA cycle, in asthenozoospermia. In the current study, immunofluorescence staining localized ACO2 to the human sperm mid-piece. By immunoblotting, we demonstrated that the level of ACO2 protein in asthenozoospermic samples was significantly decreased compared with that in normal fertile men. Importantly, we first observed that co-incubation of isocitrate with low motile sperm suspensions significantly improved sperm motility, which might be due to elevated intracellular ATP. The improvement of the sperm motility by isocitrate may have important clinical implications in the treatment of asthenozoospermia and certainly warrants further investigation.