Avian gametes present specific features related to their internal long-term mode of fertilization. Among other central actors of energetic metabolism control, it has been suspected that 5'-AMP-activated protein kinase (AMPK) influences sperm functions and thus plays a key role in fertilization success. In the present work, we studied AMPK localization and function in chicken sperm incubated in vitro. Effects of the pharmacological AMPK activators (AICAR, metformin) and the AMPK inhibitor compound C were assessed by evaluating AMPKalpha (Thr(172)) phosphorylation (by Western blotting), semen quality (by viability, motility, and ability to perform acrosome reaction), and energetic metabolism indicators (lactate, ATP). Localization of AMPK in subcellular sperm compartments was evaluated by immunocytochemistry. Total AMPK was found in all compartments except for the nucleus, but the phosphorylated form phospho-Thr(172) -AMPK was essentially localized in the flagellum and acrosome. AMPK activators significantly improved AMPK phosphorylation, sperm motility (increased by 40% motile, 90% progressive, and 60% rapid sperm), acrosome reaction and lactate production (increased by 40%) and viability. The AMPK inhibitor significantly reduced AMPK phosphorylation and percentages of motility (decrease by 25%), progressive energy (decrease by 35%), and rapid sperm (decreased by 30%), acrosome reaction, lactate production, and ATP release. The two activators differed in their effect on ATP concentration: AICAR stimulated ATP formation, whereas metformin did not. Our results indicate that AMPK plays a key role in the regulation of chicken sperm functions and metabolism. This action differs from that suggested in mammals, mainly by its crucial involvement in the acrosome reaction process.