We hypothesize that whole microalga biomass is a natural rumen-protected source of eicosapentaenoic acid (EPA, 20:5n-3) for ruminants. To test our hypothesis, we studied the ruminal biohydrogenation of EPA from two microalgae, Nannochloropsis oceanica and Phaeodactylum tricornutum using in vitro incubations with rumen fluid. A total mixed ration was incubated with: no EPA (control), EPA as free-fatty acid, N. oceanica spray-dried (SD), N. oceanica freeze-dried (FD), or P. tricornutum FD. The kinetics of EPA disappearance and of products formed during the 24 hours of incubation were evaluated, and complemented by deuterated-EPA incubation. Results showed that EPA metabolism from the N. oceanica was remarkably reduced compared with the P. tricornutum and free-EPA, and this reduction was even more effective with the N. oceanica FD. Our data also indicates that neither feed dry matter disappearance nor rumen microbial markers (branched-chain fatty acids and dimethyl acetals) were affected by EPA-sources. We reported for the first time the kinetics of EPA biohydrogenation class products and the unequivocal formation of 20:0 from EPA. Overall, N. oceanica shows a strong potential to be used as a natural dietary source of EPA to ruminants, nevertheless further studies are needed to verify its protection in vivo.