Defensins in innate immunity are known to play critical roles to protect the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between defensins and mycoplasmas. Human beta defensin (hBD)-2 and hBD-3, but not hBD-1, were found to exert strikingly antimicrobial activity against Mycoplasma pneumoniae. To elucidate the role of defensins in M. pneumoniae infection, a human pulmonary squamous cell line EBC-1 was stimulated with M. pneumoniae and interleukin (IL)-1 beta. hBD-2 was markedly upregulated by IL-1 beta as well as M. pneumoniae, but neither hBD-1 nor hBD-3 was apparently upregulated. Thus, the results suggest that inducible hBD-2 would play a critical role in the protection of M. pneumoniae infection.