Liquid storage of spermatozoa is important for artificial insemination and herd genetic breeding. However, the extended time of storage inducing the rapid decline in spermatozoa quality limits the development of this technology. The molecular mechanisms underlying liquid storage of spermatozoa remain largely unexplored. In this study, the effects of liquid storage on functional quality of spermatozoa were assessed in goat (Capra hircus). The time-dependent decline in spermatozoa motility showed a strong correlation with the significant increase in apoptosis. Moreover, apoptosis-related ultrastructural changes were observed, especially the defects in mitochondria. A significant decrease in mitochondrial membrane potential and changes in the expression of mitochondrial apoptosis-related proteins indicated mitochondrial dysfunction and mitochondrial apoptotic pathway activation. Notably, the abnormally high level of reactive oxygen species (ROS) caused by liquid storage resulted in oxidative damage to mitochondria and accelerated mitochondria-dependent apoptosis, as demonstrated by the addition of ROS scavenger N-acetylcysteine. Furthermore, critical differentially expressed proteins involved in mitochondria-dependent apoptosis and antioxidant defense were identified and profiled by quantitative proteomic analysis, facilitating the understanding of molecular regulation of ROS-induced mitochondria-dependent apoptosis. These outcomes provide insights into the mechanisms underlying liquid storage of goat spermatozoa and enhance the progress of semen storage technology.