The development of a universal influenza vaccine has become a major effort to combat the high mutation rate of influenza. To explore the use of the highly conserved stem region of hemagglutinin (HA) as a universal vaccine， we produced HA-stem-based protein using yeast expression systems. The glycosylation effects on the immunogenicity and protection activities were investigated. The yield of the A/Brisbane/59/2007 HA stem produced from Pichia pastoris reached 100 mg/l. The immunogenicity of HA stem proteins in various glycoforms was further investigated and compared. All glycoforms of the HA stem protein can induce cross-reactive antibody responses， antibody-dependent cellular cytotoxicity (ADCC)-mediated protection as well as T-cell responses， with broad protection in mice. The monoglycosylated form of the A/Brisbane/59/2007 HA stem produced in yeast， together with the glycolipid C34 as the adjuvant， can elicit greater ADCC responses， better neutralizing activities against heterologous strains， and broader protection in mice.