Glycine decarboxylase (GLDC) was recently described as a critical enzyme of tumor-initiating cells and, thus, a driver of tumorigenesis in lung non-small cell cancer (NSCC). It is important in metabolism under hypoxic conditions. Hypoxia-inducible factor 1-alpha (HIF-1α) is the unique subunit that determines HIF system activity, thereby regulating the adverse effects of hypoxia on cancer outcome. We examined the expression and prognostic significance of GLDC and HIF-1α in primary resected stage I/II NSCC. Immunohistochemistry for GLDC and HIF-1α was validated on two lung NSCC cell lines (A549, NCI-H460) and evaluated on a tissue microarray with 428 lung NSCC: 184 adenocarcinomas, 211 squamous cell carcinomas, and 33 large cell carcinomas (LCC). The results were correlated with clinico-pathological parameters. High levels of GLDC expression were detected in 33/428 cases (7.7%). HIF-1α was expressed in 71 (16.6%) cases and more frequently in squamous cell carcinoma (p < 0.001). Significantly longer survival was seen in younger patients (p = 0.007), patients with non-LCC histology (p = 0.006), lower primary tumor category (p = 0.002), and Union for International Cancer Control (UICC) stage (p = 0.001). Both GLDC and HIF-1α were significantly associated with worse tumor-related survival (p = 0.013, p = 0.021, respectively), although not independent from each other in multivariate models. The combination of low-GLDC/negative HIF-1α expression was significantly prognostic for longer survival (p = 0.002) and emerged as an independent prognostic factor in multivariate analysis (p = 0.007, HR 2.052), next to UICC stage and age. We show that the combination of GLDC and HIF-1α expression is an independent prognostic factor in early-stage NSCC. Our results will assist future development of therapeutic approaches targeting GLDC or exploiting tumor hypoxia.