Rotavirus nonstructural glycoprotein, NSP4 has been proposed as the first viral enterotoxin capable of inducing diarrhea and a target for vaccine development. In order to study biological role of NSP4, a cDNA from human rotavirus (Wa strain) RNA segment 10 was amplified by PCR, using two specific and cloned into cloning vector pBS-KS(+) and subsequently into pQE-30 expression plasmid. Expression of NSP4 was demonstrated by SDS-PAGE,Western blot and ELISA using polyclonal antibody against NSP4 from SA11 infected BSC1 cells. The recombinant protein was purified by an affinity chromatography on Nickle NTA-Agarose column (Qiagen). A polyclonal antiserum against purified recombinant NSP4 was raised in Rabbit; which was reacted with NSP4 in BSC1 cells infected with SA11 rotavirus. Intraperitoneally inoculation of NSP4 caused diarrhea in BALB/c suckling mice indicating its biological activity. Intraperitoneal and oral inoculation of NSP4 antiserum significantly decreased diarrhea disease. These results indicated successful expression and purification of the full-length biologically active, NSP4 of human rotavirus Wa strain in E. coli and showed that antibody against it was able to protect against simian rotavirus diarrhea in neonatal mice.