Cyanobacteria have massively contributed to carbonate deposition over the geological 27 history. They are traditionally thought to biomineralize CaCO 3 extracellularly as an 28 indirect byproduct of photosynthesis. However, the recent discovery of freshwater 29 cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges 30 this view. Despite the geochemical interest of such a biomineralization process, its 31 molecular mechanisms and evolutionary history remain elusive. Here, using comparative 32 genomics, we identify a new gene (ccyA) and protein family (calcyanin) possibly associated 33 with cyanobacterial iACC biomineralization. Proteins of the calcyanin family are 34 composed of a conserved C-terminal domain, which likely adopts an original fold, and a 35 variable N-terminal domain whose structure allows differentiating 4 major types among 36 the 35 known calcyanin homologs. Calcyanin lacks detectable full-length homologs with 37 known function. The overexpression of ccyA in iACC-lacking cyanobacteria resulted in 38 an increased intracellular Ca content. Moreover, ccyA presence was correlated and/or co-39 localized with genes involved in Ca or HCO 3transport and homeostasis, supporting the 40 hypothesis of a functional role of calcyanin in iACC biomineralization. Whatever its 41 function, ccyA appears as diagnostic of intracellular calcification in cyanobacteria. By 42 searching for ccyA in publicly available genomes, we identified 13 additional 43 cyanobacterial strains forming iACC, as confirmed by microscopy. This extends our 44 knowledge about the phylogenetic and environmental distribution of cyanobacterial 45 iACC biomineralization, especially with the detection of multicellular genera as well as a 46 marine species. Moreover, ccyA was probably present in ancient cyanobacteria, with 47 independent losses in various lineages that resulted in a broad but patchy distribution 48 across modern cyanobacteria.