To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro−18→Leu−18; m2, Tyr−13→Leu−13; m3, Ser−9→Leu−9; m4, Asn−5→Pro−5) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-β-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an α-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser−9, in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the α-helix of GSP, CMCase was less efficiently secreted.