Aquaporins (AQPs) are members of the major intrinsic family of proteins (MIPs) that allow pathways for water flux across cell membranes. AQPs are found across different organisms at all levels of life. To date 13 mammalian members of this family have been identified, AQP0-AQP12. AQP1 is a water channel, and under permissive conditions, also functions as a nonselective monovalent cation channel activated by intracellular cGMP. AQP1 expression facilitates cancer cell migration and spread in various cancer types including glioblastoma. Wild-type human AQP1 channels expressed in Xenopus laevis oocytes were characterized by two-electrode voltage clamp and optical osmotic swelling analyses. Cell migration was performed using wound closure assay in presence of a mitotic inhibitor to distinguish motility from proliferation. The current study has characterized a series of bumetanide derivatives for blocking effects on the ion channel function of AQP1, independent of the water channel permeability. The compound AqB011 imposed the most potent block of the AQP1 ion channel (IC50 value of 15uM) but it had no effect on the water channel activity. The order of potency was AqB001 ≤ AqB006 < AqB050 < AqB007 < AqB011. These results were consistent with in silico models predicting energetically favoured binding of the compound to the channel. AqB001 had the lowest while AqB011 possessed the highest apparent binding affinity to the channel, with likely site of interaction of AqB011 being at loop D gating domain. Furthermore AqB011 was tested on AQP1 expressing cancer cell lines and found to significantly reduce the migration ability of the cancer cells. Therefore inhibition of Aquaporin 1 could an important new therapeutic approach for reducing cancer metastasis.