Abstract Bacterial chromosome replication is regulated from a single replication origin ( ori ) that receives cell cycle signals. Following replication, bacteria often use the parABS partition system with a centromere-like parS locus to place the chromosomes into the daughter cells. Our knowledge of cell cycle regulation is incomplete and we searched for novel regulators of chromosome replication. Here we show that in the cell cycle model Caulobacter crescentus a novel DNA-binding protein promotes both the initiation of chromosome replication and the earliest step of chromosome partitioning. We used biochemical fractionation to identify a protein (OpaA) that preferentially binds to mutated ori DNA that also increases ori -plasmid replication in vivo . OpaA represents a previously unknown class of DNA-binding proteins. opaA gene expression is essential and sufficient OpaA levels are required for the correct timing of chromosome replication. Whole genome ChIP-seq identified the genomic binding sites for OpaA, with the strongest associations at the parABS locus near ori . Using molecular-genetic and fluorescence microscopy experiments, we showed that OpaA also promotes the first step of chromosome partitioning, the initial separation of the duplicated parS loci following ori replication. This separation occurs before the parABS mechanism and it coincides with the regulatory step that splits the symmetry of the chromosomes so that they are placed at distinct cell-poles which develop into replicating and non-replicating cell-types. We propose that OpaA coordinates replication with the poorly understood mechanism of early chromosome separation. opaA lethal suppressor and antibiotic experiments argue that future studies be focused on the mechanistic roles for transcription and translation at this critical step of the cell cycle. Author Summary Like all organisms, bacteria must replicate their chromosomes and move them into the newly dividing cells. Eukaryotes use non-overlapping phases, first for chromosome replication (S-phase) followed by mitosis (M-phase) when the completely duplicated chromosomes are separated. However, bacteria combine both phases so chromosome replication and chromosome separation (termed chromosome “partitioning”) overlap. In many bacteria, including Caulobacter crescentus , chromosome replication initiates from a single replication origin ( ori ) and the first duplicated regions of the chromosome immediately begin “partitioning” towards the cell poles long before the whole chromosome has finished replication. This partitioning movement uses the centromere-like DNA called “parS” that is located near the ori . Here we identify a completely novel type of DNA-binding protein called OpaA and we show that it acts at both ori and par S. The timing and coordination of overlapping chromosome replication and partitioning phases is a special regulatory problem for bacteria. We further demonstrate that OpaA is selectively required for the initiation of chromosome replication at ori and likewise that OpaA is selectively required for the initial partitioning of par S. Therefore, we propose that OpaA is a novel regulator that coordinates chromosome replication with the poorly understood mechanism of early chromosome separation.