Serine and glycine give rise to important building blocks in proliferating cells. Both amino acids are either synthesized de novo or taken up from the extracellular space. In lung cancer, serine synthesis gene expression is variable, yet, expression of the initial enzyme, phosphoglycerate dehydrogenase (PHGDH), was found to be associated with poor prognosis. While the contribution of de novo synthesis to serine pools has been shown to be enhanced by serine starvation, the impact of glucose deprivation, a commonly found condition in solid cancers is poorly understood. Here, we utilized a stable isotopic tracing approach to assess serine and glycine de novo synthesis and uptake in different lung cancer cell lines and normal bronchial epithelial cells in variable serine, glycine, and glucose conditions. Under low glucose supplementation (0.2 mM, 3–5% of normal plasma levels), serine de novo synthesis was maintained or even activated. As previously reported, also gluconeogenesis supplied carbons from glutamine to serine and glycine under these conditions. Unexpectedly, low glucose treatment consistently enhanced serine to glycine conversion, along with an up-regulation of the mitochondrial one-carbon metabolism enzymes, serine hydroxymethyltransferase (SHMT2) and methylenetetrahydrofolate dehydrogenase (MTHFD2). The relative contribution of de novo synthesis greatly increased in low serine/glycine conditions. In bronchial epithelial cells, adaptations occurred in a similar fashion as in cancer cells, but serine synthesis and serine to glycine conversion, as assessed by label enrichments and gene expression levels, were generally lower than in (PHGDH positive) cancer cells. In summary, we found a variable contribution of glucose or non-glucose carbon sources to serine and glycine and a high adaptability of the downstream one-carbon metabolism pathway to variable glucose supply.