Staphylococcal enterotoxins (SEs) are powerful superantigenic toxins produced by Staphylococcus aureus (S. aureus). They can cause food poisoning and toxic shock. However, their impact on bovine mammary epithelial cells (bMECs) is still unknown. In this study, the distribution of SE genes was evaluated in 116 S. aureus isolates from bovine mastitis, and the most prevalent genes were seh (36.2%), followed by sei (12.1%), seg (11.2%), ser (4.3%), sec (3.4%), sea (2.6%) and sed (1.7%). To better understand the effect of staphylococcal enterotoxin H (SEH) on bMECs, the seh gene was cloned and inserted into the prokaryotic expression vector, pET28a, and transformed into Escherichia coli BL21 (DE3). The recombinant staphylococcal enterotoxin H (rSEH) was expressed and purified as soluble protein. Bioactivity analysis showed that rSEH possessed the activity of stimulating lymphocytes proliferation. The XTT assay showed that 100 μg/mL of rSEH produced the cytotoxic effect on bMECs, and fluorescence microscopy and flow cytometry analysis revealed that a certain dose of rSEH is effective at inducing bMECs apoptosis in vitro. This indicates that SEs can directly lead to cellular apoptosis of bMECs in bovine mastitis associated with S. aureus.