The role of amifostine (AMF) in goat semen preservation and its modulation of mitochondrial oxidative phosphorylation were investigated by supplementing graded concentrations into hypothermic extenders and integrating sperm quality assessments, untargeted proteomics, metabolomics, and artificial insemination to evaluate functional, metabolic, and signaling alterations. AMF at 15 μg/mL provided optimal protection by maintaining sperm motility, plasma membrane and acrosome integrity, while attenuating reactive oxygen species (ROS) and malondialdehyde (MDA) levels and enhancing antioxidant enzyme activities, mitochondrial membrane potential (MMP), and sperm ATP content. Consequently, the pregnancy rate reached 72.00% after 5-day storage, comparable to fresh semen (76.20%) and markedly higher than the control (53.48%). Proteomic analysis identified 44 differentially expressed proteins, including ubiquinone oxidoreductase subunit A10 (NDUFA10), electron transfer flavoprotein dehydrogenase (ETFDH), hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB), and citrate synthase (CS), enriched in oxidative phosphorylation (OXPHOS), fatty acid β-oxidation, and the tricarboxylic acid cycle (TCA) cycle. Metabolomic profiling revealed 494 differential metabolites, primarily linked to carbon metabolism and the TCA cycle, with elevated α-ketoglutarate (α-KG). Integrative analysis suggested coordinated regulation of glycolysis, fatty acid oxidation, and OXPHOS. Mechanistically, AMF increased p-AMPK and decreased p-GSK3α, indicating activation of the AMPK/GSK3α axis. AMF improves hypothermic semen preservation and fertility through AMPK/GSK3α-driven reprogramming of mitochondrial oxidative metabolism, providing a multi-omics framework to optimize liquid semen storage and artificial insemination (AI).