Biselyngbyasides (BLSs), macrolides from a marine cyanobacterium, are cytotoxic natural products whose target molecule is unknown. Here we report that BLSs are high affinity (K-i similar to 10 nM) inhibitors of Ca2+-pumps with a unique binding mode. The crystal structures of the Ca2+-pump in complex with BLSs at 3.2-3.5 angstrom-resolution show that BLSs bind to the pump near the cytoplasmic surface of the transmembrane region. The crystal structures and activity measurement of BLS analogs allow us to identify the structural features that confer high potency to BLSs as inhibitors of the pump. (C) 2015 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.