Caulobacter crescentus is a Gram-negative bacteria that divides asymmetrically. In this organism, the formation of the FtsZ ring (Z-ring) at the correct position in the cell requires the formation of a bipolar gradient of the ATPase MipZ. The chromosome partitioning protein ParB, an interaction partner of MipZ, is anchored at the cell poles by the scaffolding protein PopZ. In this study, we have uncovered the inhibitory mode of action of MipZ on FtsZ polymerization and identified the interaction regions of MipZ with its three interaction partners, ParB, FtsZ and the chromosomal DNA. We also purified PopZ from soluble fractions and provided relevant data related to its secondary structure composition and its assembly into higher-order structures.