For decades Selenium (Se) research has been focused on the identification of active metabolites, which are crucial for Se chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include: Increased formation of reactive oxygen species (ROS), induction of DNA damage, triggering of apoptosis and the inhibition of angiogenesis. Here, we revealed that CH3SeH modulates cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways, which occur early during malignant transformation, and enables recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional level: CH3SeH induced the transcription of MICA/B and ULBP2 mRNA, however, the induction of cell-surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH -regulation of NKG2D ligands. A balanced cell-surface expression of NKG2D ligands is considered as an innate barrier against tumor development. Our work therefore indicates that the application of selenium compounds, which are metabolized to CH3SeH, could improve NKG2D-based immune therapy.