Prolonged hyperoxia exposure leads to inflammation and acute lung injury. Since hyperoxia activates nuclear factor kappa B (NF-κB) and proinflammatory mediators in lung fibroblasts and murine lungs, and proinflammatory cytokines upregulate Tn (N-acetyl-d-galactosamine-O-serine/threonine) expression in human gingival fibroblasts. We hypothesized connections exist between Tn expression and inflammation regulation. Thus, we immunized adult mice with Tn antigen to examine whether Tn vaccine can protect against hyperoxia-induced lung injury by inhibiting NF-κB activity and cytokine expression through the action of anti-Tn antibodies. Five-week-old female C57BL/6NCrlBltw mice were subcutaneously immunized with Tn antigen four times at biweekly intervals, and one additional immunization was performed at 1 week after the fourth immunization. Four days after the last immunization, mice were exposed to room air (RA) or hyperoxia (100% O2) for up to 96 h. Four study groups were examined: carrier protein + RA (n = 6), Tn vaccine + RA (n = 6), carrier protein + O2 (n = 6), and Tn vaccine + O2 (n = 5). We observed that hyperoxia exposure reduced body weight, increased alveolar protein and cytokine (interleukin-6 and tumor necrosis factor-α) levels, increased mean linear intercept (MLI) values and lung injury scores, and increased lung NF-κB activity. By contrast, Tn immunization increased serum anti-Tn antibody titers and reduced the cytokine levels, MLI values, and lung injury scores. Furthermore, the alleviation of lung injury was accompanied by a reduction in NF-κB activity. Therefore, we proposed that Tn immunization attenuates hyperoxia-induced lung injury in adult mice by inhibiting the NF-κB activity.