Lung adenocarcinoma is characterised by an upregulation of S100P, which mediates its function intracellularly but also extracellularly. Recent studies suggest that extracellular S100P contributes to tumour development following interaction with the receptor for advanced glycation end-products (RAGE). As RAGE is highly downregulated in lung cancer, one might speculate that S100P supports tumorigenesis via other pathways. Here, we showed that S100P primarily localises in the cytoplasmic and nuclear region as determined for lung adenocarcinoma specimens (immunohistochemistry) and H358 lung adenocarcinoma cells stably overexpressing S100P (GFP-S100P; fluorescence microscopy). S100P overexpression in H358 cells induced a more frequent formation of tumour colonies in vitro (soft agar assay). However, the S100P-overexpressing cell colonies showed a smaller colony size. This observation was supported by proliferation assays demonstrating a reduced proliferation per increasing cell density of the H358 cells overexpressing S100P. Migration of S100P-overexpressing H358 cells was diminished (transwell migration and in vitro wound scratch assays). Inhibiting DNA methylation with 5-aza-2'deoxycytidine enhanced the mRNA expression of S100P, whereas high S100P levels following overexpression suppressed the mRNA expression of endogenous S100P. In this regard, an upregulation of S100P in lung adenocarcinomas was only determined for early/T1 stage but not more advanced/T2 stage tumours compared with normal lung tissues. Thus, S100P induction may be considered an important step in the initial stage of lung adenocarcinomas, whereas its downregulation in advanced stages seems to be important for tumour progression in which DNA methylation and/or feedback transcription processes play a critical role.