Devastating outbreaks of porcine epidemic diarrhea (PED) started in China in late 2010 and rapidly spread to North America and Asia causing severe diarrhea and high mortality in neonatal piglets， indicating that a new generation of vaccine against porcine epidemic diarrhea virus (PEDV) is urgently needed. In the present study， to mimic the native spike (S) glycoprotein， a stable cell line producing the trimeric ectodomain of S glycoprotein of the PEDV Pintung-52 (PEDV-PT) strain was successfully established by incorporating T4 bacteriophage foldon sequence of fibritin trimerization domains at the C-terminal end and replacing the signal peptide of S protein with the tissue plasminogen activator signal peptide sequence at the N terminal end. The trimeric structure， bio-reactivity to PEDV-specific antibodies， and the N-glycosylation level of the recombinant S protein were characterized. To induce systemic and mucosal immunity， conventional 5-week-old piglets were immunized with the trimeric S glycoprotein combined with the B subunit of Escherichia coli heat-labile enterotoxin (LTB) by the intramuscular (IM) route. As compared with the control group， all piglets in the S protein-LTB immunized (IM PEDV S-LTB) group generated systemic PEDV S-specific IgG and neutralizing antibody in blood but a low level of fecal PEDV-specific IgA and limited protection against challenge of PEDV-PT strain. Our results suggest that the recombinant PEDV trimeric S glycoprotein could be a potential subunit vaccine candidate against PEDV， but IM immunization with LTB as the adjuvant provided insufficient protection. The development of a vaccine regimen for inducing mucosal immunity is an important task for generating a successful subunit vaccine against PEDVs.