Bacterial cell division relies on the formation and contraction of the Z-ring, coordinated and regulated by a dynamic protein complex called divisome. Here, we show that ZapB, a newly identified cell division factor in Escherichia coli, is recruited to the division site in the early stages of Z-ring assembly by ZapA. Inactivation or overproduction of ZapA caused ZapB delocalization and diffusion. Bacterial two-hybrid and in vitro assays showed that ZapB interacts directly with ZapA and, through it, with FtsZ and that the three proteins together can form a highmolecular- weight complex. Furthermore, during the cell cycle ZapB closely followed FtsZ dynamic localization but interestingly, using high-resolution 3D reconstruction microscopy, we found that it formed a ring located on the inside of the Z-ring, consistently in all cells in consecutive cell division events. Only in the absence of the bacterial actin homologue MreB, ZapB was not able to constrict ahead of FtsZ and instead co-localized seemingly perfectly with the Z-ring. Morphological analysis of cells carrying a zapB deletion and the ftsZ84 allele exhibited a synthetic detrimental phenotype and cell division defects. A model in which ZapB further increases the lateral association of FtsZ filaments by cross-linking ZapA molecules bound to adjacent FtsZ filaments is supported by light scattering assays and analysis of structures formed by FtsZ-ZapA-ZapB using electron microscopy. Surprisingly, ZapB seemed to be active in cell division in the absence of ZapA raising the possibility that ZapB might have a secondary ZapA-independent function.