Surfactant proteins play important roles in lung surfactant function and innate immunity. The DNA methylation state of 11 CpG sites of surfactant protein (SP)-A1, -B, -C, and -D was determined using universal bead arrays. A total of 90 cancerous and non-cancerous tissues from 23 patients with adenocarcinoma and 22 with squamous cell carcinoma were studied. These were divided into a training set and a testing set. The results indicate that DNA methylation profiling of these CpGs is associated with lung cancer. Four CpG sites, SP-A1_370, SP-A1_1080, SP-D_1170, and SP-D_1370, were hypomethylated in cancer and were significantly associated with both adenocarcinoma and squamous cell carcinoma, indicating that they have the potential to be used as biomarkers for lung cancer diagnosis and treatment. Normal lung tissues with a higher level of unmethylated SP-A1_1468 and SP-D_1170 CpG exhibited a higher level of SP-A1 and SP-D gene transcripts indicating that CpG methylation may play a role in gene expression. When the non-cancerous tissues were compared to cancerous tissues in patients with adenocarcinoma, the methylation profile results of these 46 samples (23 cancerous and 23 non-cancerous) could be clustered into 4 groups by agglomerative nesting. The percentage of tumor samples in each group was 0, 58, 91, and 100, respectively. A similar pattern was observed in squamous cell carcinoma patients. We speculate that SP-A1 and SP-D are subject to methylation/demethylation regulatory mechanisms and are involved in lung cancer pathogenesis by virtue of their function in innate host defense and/or regulation of inflammation.