Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer related deaths worldwide. The prognosis of lung cancer patients is very poor with a 5-year survival rate that is only ~15 % in advanced stages. The early devolopment of metastasis is one of the main reasons for this poor prognosis. Metastasis is a complex phenomen in which the process of extravasation is of enormous importance. In principal, extravasation can be devided into the processes of adhesion to endotheial cells and transmigration through the endothelial layer. In our study, we were investigating whether the Ca2+ activated K+ channel KCa3.1 plays a role in cell-cell adhesion of A549 NSCLC cells to HMEC-1 endothelilal cells, and in the transmigration process of A549 cells. Applying single force spectroscopy, we estimated the adhesion force [nN] between A549 and HMEC-1 cells. The role of KCa3.1 channels was assessed pharmacologically (KCa3.1 channel blocker senicapoc) or by silencing of KCa3.1 in A549 or HMEC-cells. In the presence of senicapoc the adhesion force increased to 49% (0.85 ± 0.03 nN in senicapoc vs 0.43 ± 0.02 nN in DMSO). Silencing of KCa3.1 in A549 cells increased the adhesion force from 0.48 ± 0.02 nN (in control transfected cells) to 0.62 ± 0.03 nN, in HMEC-1 cells from 0.54 ± 0.04 nN to 0.82 ± 0.05 nN. Western blotting (N = 3) and immunofluorescence staining (N = 3) reveals that the elevated adhesion force is due to increased expression of ICAM-1 in both cell lines when KCa3.1 channels are silenced. Appliying an anti-ICAM-1 blocking antibody abolishes the KCa3.1-dependent strengthening of cell-cell adhesion (N = 7; p < 0.05; One-Way ANOVA). Furthermore, using an in vitro approach for transendothelial migration, we found that senicapoc inhibits transendothelial migration of A549 cells by 50% (N = 6; p < 0.05; students t-test). This inhibiton in transendothelial migration was not found when KCa3.1 channels were silenced in A549 cells. The latter finding suggests that transendothelial migration depends predominantly on KCa3.1 channels in endothelial cells. In conclusion, our findings indicate that KCa3.1 channels regulate ICAM-1 dependent cell-cell adhesion between A549 lung cancer and HMEC-1 endothelial cells and that also affects the transmigration step in the metastatic process.