Identification of novel risk factors that are critical to the initiation of lung cancer will be key for its prevention. Recently, it has been reported that glycine dehydrogenase (GLDC) can drive the formation of lung cancer initiating cells. However, there have been no perspective studies on the association between circulating GLDC and lung cancer until now. To identify whether serum GLDC is a risk factor for lung cancer, the present study conducted a nested case‑control study within a Chinese cohort. Using ELISAs, serum GLDC was measured in 300 case subjects, who were subsequently diagnosed with lung cancer during follow‑up, and in 600 matched healthy controls. The results revealed that serum GLDC was associated with increased lung cancer risk [odds ratio=1.48; 95% confidence intervals (1.01‑2.04)]. Spearman correlation was employed to analyze the associations between age, body mass index, years of smoking and the serum concentration of GLDC. It was demonstrated that years of smoking was associated with serum GLDC (spearman's correlation, ρ=0.81) in patients with lung cancer. However, the association was attenuated in the serum of matched controls (ρ=0.48). In addition, overexpression of GLDC protein contributed to malignant transformation and inhibited microRNA (miR)‑29 family expression in normal human bronchial epithelial (NHBE) cells. Aberrant methylation of tumor suppressive gene (TSG) is an early event in the development of lung cancer, which is controlled by DNA methyltransferases (DNMTs). The present study demonstrated that GLDC promoted the expression of DNMT proteins; however, the miR‑29 family inhibited their expression in NHBE cells. Thus, it was concluded that elevated serum GLDC may increase lung cancer risk, and that smoking, GLDC, the miR‑29 family and DNMT signaling pathways may serve an important role in early malignant transformation during the development of lung cancer.