Mucosal vaccines are considered to be a promising strategy for preventing upper airway infections. As immunization routes to induce mucosal immune responses, oral, intranasal and sublingual immunizations have been proposed for clinical application. Recently, it was reported that transcutaneous immunization (TC) is also capable of inducing mucosal as well as systemic immune responses. However, it remains unclear which route is most effective for inducing mucosal immune responses in upper respiratory airways. In this study, the mucosal immune responses against phosphorylcholine (PC), a structural component of a wide variety of Gram-positive and -negative bacteria, were investigated after intranasal, sublingual, or TC. Female BALB/c mice were immunized intranasally, sublingually, or transcutaneously with PC and cholera toxin. For the TC, the dorsal region was shaved and a cotton patch soaked with the antigen was attached. Nasal wash, saliva and serum samples were collected at 7 days after the final immunization, and examined for their PC-specific antibody activities using ELISA. Phosphorylcholine-specific antibodies in serum and PC-specific IgA in nasal washes and saliva were detected in mice after intranasal and sublingual immunization with PC. Although salivary IgA was higher following intranasal immunization, nasal wash IgA was significantly higher after sublingual immunization. Furthermore, significantly increased IgA in vaginal washes and remarkably decreased serum IgE production were observed after sublingual immunization. TC increased PC-specific IgA in faecal samples, but not in saliva. The PC antigen can induce mucosal as well as systemic immune responses when administered via intranasal, sublingual and transcutaneous routes. Sublingual immunization is superior to intranasal immunization in terms of safety for inducing mucosal immune responses. Although TC might be an alternative way to induce mucosal immune responses, further investigation is needed for its application as a vaccine route to prevent upper airway infection.