FtsZ is a tubulin-like protein present in most bacteria, that self-assembles into filaments forming a structure known as Z-ring in the cells. Following formation, the Z- ring recruits a protein macrocomplex, the divisome, which is responsible by the division septum synthesis, resulting in two daughter cells. Many modulators interact directly to FtsZ, regulating its polymerization and controlling the time and place of the Z-ring formation. MciZ is a 40-amino-acid peptide that is expressed during sporulation in Bacillus subtilis and inhibits the formation of the Z-ring in the mother-cell. The aim of this work was to study the interaction between FtsZ and MciZ proteins, and to investigate the mechanisms involved in FtsZ inhibition by MciZ. Applying a genetic screening, using an ftsZ mutant library, we identified 13 mutations on ftsZ that conferred resistance to MciZ overexpression in vivo. Seven of them were able to grow either in the presence or absence of MciZ overexpression, and the other six showed to be dependent on it. With the co-expression and co-purification of the FtsZ:MciZ complex, we observed all mutant proteins still interact with MciZ in vitro. Estimated Kd for the interaction was 150±50nM, and it was demonstrated that MciZ does not bind to FtsZ CTP (C-Terminal Peptide), nor does it compete with GTP for the same binding site. Using truncated versions of MciZ, it was determined that its N-terminal (residues 1 to 27) is sufficient for the inhibition. Based on the tridimensional structure of MciZ (NMR) and of the FtsZ:MciZ complex (x- ray crystallography), it was determined that MciZ is an unstructured peptide that assumes a tertiary structure by interacting with FtsZ α-helix H10 and β-sheet S9 through its α-helix H2 and β-sheet B2. MciZ was able to reduce the size of FtsZ protofilaments in a substoichiometric manner, generating smaller fragmented filaments. 1:10 ratios of MciZ:FtsZ were sufficient to completely extinguish the Z-ring, thus confirming the substoichiometric inhibition in vivo as well. The inhibition of FtsZ polymerization by the FtsZ-MciZ fusion and the FtsZ depolymerization kinetics induced by MciZ proved that MciZ is not a simple sequesterer. Fluorescent dyeing of MciZ suggests the peptide is able to interact with the Z-ring in vivo, as well as decorate FtsZ bundles in vivo, forming localized spots frequently at the filaments' ends. Co- sedimentations with FtsZ polymers showed the presence of MciZ or of the FtsZ-MciZ fusion. Despite MciZ-induced increase in specific GTPase activity of FtsZ, the lack of GTP hydrolysis did not eliminate the substoichiometric effect of MciZ. Combined, our results show that MciZ is an FtsZ filament capper, blocking elongation at the minus end and blocking the annealing between protofilaments