E2F transcription factors are key regulators of cell proliferation that are inhibited by pRb family tumor suppressors. pRb-independent modes of E2F inhibition have also been described, but their contribution to animal development and tumor suppression is unclear. Here we show that S phase-specific destruction of Drosophila E2f1 provides a novel mechanism for cell cycle regulation. E2f1 destruction is mediated by a PCNA-interacting-protein (PIP) motif in E2f1 and the Cul4Cdt2 E3 ubiquitin ligase, and requires the Dp dimerization partner but not direct Cdk phosphorylation or Rbf1 binding. E2f1 lacking a functional PIP motif accumulates inappropriately during S phase and is more potent than wild type E2f1 at accelerating cell cycle progression and inducing apoptosis. Thus, S phase-coupled destruction is a key negative regulator of E2f1 activity. We propose that pRb-independent inhibition of E2F during S phase is an evolutionarily conserved feature of the metazoan cell cycle that is necessary for development.