The β-defensins, expressed in epithelial cells of multiple tissues including intestine, play a critical role in the mammalian innate immunity. However, it is little known about the role of functional nutrients in the regulation of porcine β-defensins' expressions in intestinal epithelial cells. The present study was conducted to determine the hypothesis that zinc and L-isoleucine regulate the expressions of porcine β-defensins in IPEC-J2 cells. Cells were cultured in DMEM/F12 medium containing supplemental 0-500 μg/mL L-isoleucine or 0-500 μmol/mL zinc sulfate that was used to increase the concentration of Zn(2+) in the medium. At 12 h after the treatment by the appropriate concentrations of L-isoleucine or Zn(2+), the mRNA and protein expressions of porcine β-defensin 1, 2 and 3 were increased (P < 0.05), and reached their maximum after treatment with 25 or 100 μmol/mL zinc sulfate and 25 or 50 μg/mL isoleucine (P < 0.05). These results suggested that both Zn(2+) and L-isoleucine could induce β-defensins' expressions in porcine intestinal epithelial cells.