DNA replication, the key determinant of cell proliferation, is a highly regulated process whose objective is to ensure that the genetic information encoded in the chromosomes is faithfully transmitted from parental to progeny cells. Failure in this regulation could lead to abnormal proliferation and genomic instability, the hallmarks of cancer cells. The initiation step in DNA replication constitutes the main target in the alteration of cell proliferation. Initiation of DNA replication requires the activation of thousands of origins of DNA replication (ORIs) which fire only once, in a pre-determined temporal order. In this review we summarize the salient features of this process, and present experimental evidence indicating that carcinogenesis and/or genomic instability may arise by a failure in the regulation of the initiation step of DNA replication.