The formation of the pre-RC is an important event during DNA replication initiation. It occurs from late M phase until early G1 phase and is required to license the replication origin before the binding of other initiation factors. Cdt1, one of the pre-RC components, has been shown to be a target of regulation after DNA damage in different model organisms. Previous work from our group identified a novel G1/S checkpoint in fission yeast, and this checkpoint is dependent on the Gcn2 kinase which has a known role of leading to downregulation of global translation under amino acid starvation. The activation of this checkpoint after UVC irradiation in early G1 phase results in delayed pre-RC formation and consequently, a delayed G1/S transition. However, the underlying connection between the delayed pre-RC formation and the regulation of Cdt1 in response to UVC irradiation in early G1 phase is not yet clear. The work carried out in this study is aimed to investigate the impact of changes in Cdt1 level on the kinetics of pre-RC formation in fission yeast, especially when the G1/S checkpoint is activated by UVC irradiation. We explored the expression of Cdt1 after UVC irradiation in early G1 phase and show that Cdt1 level is reduced. We also show that gcn2Δ cells with defective G1/S checkpoint have more Cdt1 than that in cells with intact G1/S checkpoint after UVC irradiation in early G1 phase. These observations lead us to speculate that the reduced Cdt1 level is limiting for pre-RC formation when the G1/S checkpoint is activated. We show that consistently, in cells overexpressing Cdt1 the increase in pre-RC formation correlates with the increase in Cdt1 level after UVC irradiation in early G1 phase. This result suggests that the mechanism of the G1/S checkpoint involves reducing the Cdt1 level, which in turn is limiting for pre-RC formation and thus leads to a delayed G1/S transition.