Effect of an extraction method on the structure of glucan and its immunostimulatory response in Fenneropenaeus indicus was investigated. Here we extracted alkali insoluble glucan (AIG) and alkali soluble glucan (ASG) from a filamentous fungi Acremonium diospyri following alkali-acid hydrolysis and the sodium hypochlorite oxidation and dimethyl sulphoxide extraction method respectively. Structural analysis showed that 85% of glucan in AIG was a (1→3)-β-d-glucan and it increased the prophenoloxidase and reactive oxygen intermediate activity when administered to F. indicus. On the other hand, ASG, which contained 93% (1→3)-α-glucan, did not induce significant immune response in shrimp. Here we report that the difference in immunostimulatory potential between AIG and ASG is due to the difference in the percentage of (1→3)-β-d-glucans present in each preparation, which varies with the method of extraction employed. Also our observations suggest that glucan can be used as a potential immunostimulant to shrimp, provided it contains (1→3)-β-d-glucan as the major fraction.