DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by a limited understanding of DNA replication in pathogenic micro-organisms. Clostridium difficile is the main cause of health-care associated diarrhea and its DNA replication machinery is virtually uncharacterized. We identified the replicative helicase (CD3657), the helicase loader ATPase (CD3654) and primase (CD1454) of C. difficile and reconstitute helicase and primase activity in vitro. We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. We find that helicase activity is dependent on the presence of primase in vitro. The trinucleotide specificity of primase, which we find to be determined by a single lysine residue, is similar to primase of the extreme thermophile Aquifex aeolicus but the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, helicase-primase interactions constitute a crucial aspect of DNA replication initiation in C. difficile and helicase loading and activation in this organism differs critically from that of the Gram-positive model organism Bacillus subtilis.