The chromosomal DNA replication in eukaryotic cells begins at replication initation sites, which are marked by the assembly of the pre-replication complexes in early G1. At the G1/S transition, recruitment of additional replication initiation proteins enables origin DNA unwinding and loading of DNA polymerases. We found that depletion of the human DNA helicase B (HDHB) inhibits the initiation of DNA replication, suggesting a role of HDHB in the beginning of the DNA synthesis. To gain insight into the function of HDHB during replication initiation, we examined the physical interactions of purified recombinant HDHB with key initiation proteins. HDHB interacts directly with two initiation factors TopBP1 and Cdc45. In addition we found that both, the N-terminus and helicase domain of HDHB bind to the N-terminus of Cdc45. Furthermore depletion of HDHB from human cells diminishes Cdc45 association with chromatin, suggesting that HDHB may facilitate Cdc45 recruitment at G1/S in human cells.