AbstractChannelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H⁺, Na⁺, K⁺ and anions have been discovered or engineered, Ca²⁺-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca²⁺ permeability and improve their Ca²⁺ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca²⁺ permeation at negative voltage and low extracellular Ca²⁺ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca²⁺ concentrations. Moreover, CapChR2 can robustly trigger Ca²⁺ signalling in hippocampal neurons. When expressed together with genetically encoded Ca²⁺ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca²⁺ entry in brain explants.