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About 20 different types of staphylococcal enterotoxins are produced by Staphylococcus aureus, in which type A is more common in food poisoning syndrome. Also staphylococcal enterotoxin A superantigen is a potent inducer of cytotoxic T lymphocyte activity and cytokine production and could stimulate T cells containing T-cell receptor beta chain domains when binding to major histocompatibility complex class II molecules. Hence, it is an important reagent in cancer immunotherapy.For the construction of pET-21a/entA cassette, the staphylococcal enterotoxin type A gene was isolated from S aureus strain HN2, cloned into pET-21a, and introduced into Escherichia coli strain BL-21(DE3). Consequently, Western blot analysis showed pET-21a/entA cassette expression inserted entA gene successfully. It is the first prompt using a pET-21a as a cloning vector for entA gene and expression of construct in BL-21(DE3). In addition, this study examined the ability of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A to activate T cells in vitro. Lymphocyte cells derived from lymph node BALB/c mice were exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin (1.10, 102,103, and 104 ng/µL) in order to evaluate the magnitude of proliferation, activation, and apoptosis of lymphocyte cells based on MTT and apoptosis assays, respectively.Our investigation showed that the function of cloned staphylococcal enterotoxin A was same as standard staphylococcal enterotoxin A, and the optimal concentration for the activation of lymphocyte cells and induction of apoptosis was 100 ng/µL and 1000 ng/µL (P < .05), respectively. Quantification of cytokines clearly showed that lymphocyte cells exposed to standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A significantly secreted higher interferon γ and tumor necrosis factor α compared to control.According to our results, the biological activity of standard staphylococcal enterotoxin A and cloned staphylococcal enterotoxin A is identical; therefore, these procedures may be approved as an efficient method to express and purify this protein in a large scale.