Respiratory Mycoplasma pneumoniae (Mp) infection is involved in several acute and chronic lung diseases including community-acquired pneumonia, asthma and chronic obstructive pulmonary disease. In the chronic disease process, recurrent respiratory bacterial infections could occur, which may result in varying degrees of symptoms and lung inflammation among patients. However, the lung immunologic differences of host responses to repeated bacterial (i.e., Mp) infections remain to be determined. In the present study, we examined cellular and humoral responses to multiple (up to 3) Mp infections in two genetically different strains of mice (BALB/c and C57BL/6). Mice were intranasally inoculated with one Mp infection, two or three Mp infections (4 weeks apart), and sacrificed on days 3, 7 and 14 after the last Mp infection. Overall, compared to C57BL/6 mice, BALB/c mice demonstrated a significantly higher degree of lung tissue inflammatory cell infiltrate, BAL cellularity, and release of pro-inflammatory cytokines (TNF-alpha, keratinocyte-derived chemokine (KC, a mouse homolog of human chemokine Gro-alpha [CXCL1], and IFN-gamma). In addition, BALB/c mice presented higher levels of serum Mp-specific IgG and IgM, but not IgA. Consistently with lung and serum data, Mp load in BAL and lung specimens was significantly higher in BALB/c mice than C57BL/6 mice. Moreover, repeated Mp infections in BALB/c, but not C57BL/6 mice, produced a greater inflammatory response than did a single Mp infection. Our results suggest that hosts with different genetic background may have different susceptibility to repeated respiratory Mp infections along with inflammatory responses.