Here, we provide evidence for the first time that CaMKKβ regulates store‐operated Ca2+ entry (SOCE) through controlling the transcription of stromal interaction molecule 1 (STIM1), a crucial activator of SOCE in endothelial cells (ECs). Surprisingly, we observed drastically reduced expression of both mRNA and protein for STIM1 in lung tissue and lung ECs of CaMKKβ knockout (CaMKKβ−/−) mice compared with wild type. Interestingly, expression of STIM2, TRPC1, TRPC4, Orai1, Orai2, and Orai3 was unaltered in CaMKKβ−/− mice. Knockdown of CaMKKβ in human lung microvascular endothelial cells (HLMVECs) also suppressed STIM1 expression. Importantly, we observed complete loss of SOCE in LECs of CaMKKβ−/− mice compared to the wild type mice. Since CaMKKβ may regulate gene expression through epigenetic mechanism, we analyzed STIM1 gene sequence and observed the presence of methylation‐prone CpG sites in 5′‐regulatory of both human and mouse STIM1 genes. DNA methylation analysis revealed hypermethylation of ST1M1 promoter in CaMKKβ−/− mice compared to WT mice. Interestingly, i.p injection of 5‐azacytidine, a methyl transferase inhibitor restored STIM1 expression in lungs of CaMKKβ−/− mice. In addition, liposome‐mediated in vivo delivery of WT‐CaMKKβ but not by K193A‐CaMKKβ plasmid restored STIM1 expression in lung ECs of CaMKKβ−/− mice. These findings demonstrate the prerequisite role of CaMKKβ in regulating STIM1 transcription through epigenetic mechanism in endothelial cells.Support or Funding InformationNational Institute of Health Grants R01 HL‐128359 and R01 GM‐117028This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.