Background: Endoreplication is a variant cell cycle which generates massive DNA replication with no features of mitosis. In addition to abnormal occurrence of endoreplication in cancer cells, it is often found in plants and many different animal organs, such as liver, placenta, and Drosophila larval tissues. In treatment with anti‑mitotic drugs, it has been shown that cancer cells may undergo endoreplication to escape apoptosis. However, the underlying mechanisms of endoreplication in normal and pathological circumstances remain obscure. Methods: The regulation and function of most physiological processes are highly conserved between the fruit fly Drosophila melanogaster and mammals. In addition, using Drosophila as a research model can largely reduce genetic redundancy issues and provide a suitable way to observe cell autonomy. To address the aforementioned questions, we use the Drosophila as an animal model to study the function of fundamental regulators in endoreplication. Results: In the present study, we demonstrated that high levels of bHLH transcription factor E protein are capable of inhibiting endoreplication in larval salivary glands. The negative regulation of E protein in endoreplication depends on the dysregulation of cell cycle regulators, including E2f1 and its target genes Cyclin E and PCNA. However, the endoreplication defects caused by E protein overexpression are independent of the Hippo tumor suppressor pathway. Conclusions: Our results reveal that endoreplication can be prevented by high levels of E protein through disrupting the oscillations of cell cycle regulators.