In the Pacific oyster, spermatozoa are characterized by a remarkably long movement phase (i.e., over 24 h) sustained by a capacity to maintain intracellular ATP level. To gain information on oxidative phosphorylation (OXPHOS) functionality during the motility phase of Pacific oyster spermatozoa, we studied: i) changes in spermatozoa mitochondrial activity (i.e., mitochondrial membrane potential, MMP) and intracellular ATP content in relation to motion parameters and ii) the involvement of OXPHOS for spermatozoa movement using CCCP. The percentage of motile spermatozoa decreased over a 24 h movement period. MMP increased steadily during the first 9 h of the movement phase and was subsequently maintained at a constant level. Conversely, spermatozoa ATP content decreased steadily during the first 9 h post activation and was maintained at this level during the following hours of the movement phase. When OXPHOS was decoupled by CCCP, the movement of spermatozoa was maintained 2 h and totally stopped after 4 h of incubation, whereas spermatozoa were still motile in the control after 4 h. Our results suggest that ATP sustaining flagellar movement of spermatozoa may partially originate from glycolysis or from mobilization of stored ATP or potential phosphagens during the first 2 h of movement as OXPHOS was decoupled by CCCP. However, OXPHOS is required to sustain the long motility phase of Pacific oyster spermatozoa. In addition, spermatozoa may hydrolyse intracellular ATP content during the early part of the movement phase, stimulating mitochondrial activity. This stimulation seems to be involved in sustaining a high ATP level until the end of the motility phase.