Aim Although it is accepted that progesterone (P) induces acrosome reaction through non-genomic regulation, it is not well known if P also affects hyperactivation of sperm. Methods Hamster spermatozoa were hyperactivated by incubation for 4 h on modified Tyrode’s albumin lactate pyruvate medium and recorded on a DVD via a charge-coupled device camera attached to a microscope with phase-contrast illumination and a small CO 2 incubator. Phosphorylation of proteins was detected by western blotting using antiphosphotyrosine antibodies. Results Sperm hyperactivation was significantly increased and accelerated by a non-genomic signal of P. Although acceleration of motility of hyperactivated sperm occurred with 10, 20 and 40 ng/mL P, the most effective concentration was 20 ng/mL. Progesterone also significantly increased 80-kDa tyrosine phosphorylation of sperm proteins. Both extracellular Ca 2+ and albumin were essential for sperm hyperactivation, and the former was also essential for maintaining sperm flagellar movement. Moreover, phospholipase C (PLC) was associated with the regulation of hyperactivation by P. Conclusion It is likely that P regulates sperm hyperactivation by a non-genomic signal in relation to tyrosine phosphorylation and PLC.