Recent studies indicated that estrogens may influence the development of non-small cell lung cancer (NSCLC). The 17-beta-hydroxysteroid dehydrogenase type 1 (HSD17B1) catalyzes the reduction of estrone (E1) to the highly potent E2. Although the significance of aromatase in an intratumoral E2 production in NSCLC is well established, the role of HSD17B1 remains largely unknown. Therefore, we investigated the expression of HSD17B1 in lung cancerous and corresponding histopathologically unchanged tissues from NSCLC patients and the association between HSD17B1 expression and clinicopathological features. Than, we examined the biological significance of HSD17B1 in NSCLC cells in vitro. We tested the impact of 5-Aza-2′-deoxycytidine (5-dAzaC) on HSD17B1 expression and activity.We used Real Time quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry to evaluate HSD17B1 expression in tissues obtained from 48 patients with NSCLC. The methylation status of the promoter region of HSD17B1 in A549 and Calu-1 cells was evaluated by bisulfite sequencing. We investigated the effect of 5-dAzaC on HSD17B1 transcript levels (by RT-qPCR) and on HSD17B1 enzyme activity by measuring the conversion of E1 to E2. The xCELLigence System was used for monitoring of cell proliferation.We found a substantial increase of HSD17B1 mRNA and protein amount in NSCLC tissues compared with histopathologically unchanged tissues in the group of male patients. An overexpression of HSD17B1 was associated with squamous cell carcinoma and with lung cancer stage 3A. We showed that 5-dAzaC induces DNA demethylation of HSD17B1 promoter, leading to increased HSD17B1 mRNA levels and protein activity in NSCLC cells. It resulted in enhanced E2 production in both cell lines and supported the proliferation of Calu-1 cells but not A549 cells.Increased expression of HSD17B1 in NSCLC may contribute to an elevated intratissue level of E2 and consequently may support the development and spread of cancer.