The thrombolytic agent DSPAα1 is currently undergoing clinical trials for the treatment of acute ischemic stroke and has shown good pharmacodynamic, pharmacokinetic and safety profiles. Here, the DSPAα1 gene, optimized for the preferred codons of yeast, was cloned into the Pichia pastoris strains GS115 and KM71. Both expression systems produced functional DSPAα1 into the broth medium under shaking flask growth conditions with the yield of about 70 mg/L, and 105 mg/L, respectively. In addition, three glycosylation minus DSPAα1 mutants, constructed by site-directed mutagenesis, were also expressed in Pichia pastoris. The mutant proteins were assayed by SDS-PAGE and fibrin degradation activities were evaluated. The secretion levels of all the mutants, especially N362Q and N117Q/N362Q, were so lower compared to the wild-type DSPAα1 that only minimal quantities of mutant protein could be recovered by purification from the culture medium. The protein specific activities from mutants (N117Q, N362Q) were less 25% than that of the wild type protein. These results imply that the N-linked carbohydrate chains (at N117 and N362) are vital for the enzymatic activity of rDSPAα1 and for its secretion from Pichia pastoris.