DGK theta protein expression levels are closely related to the development of diseases including diabetes, cancer, and neuronal disease. To investigate the transcriptional regulation of the DGK theta gene, we used CRISPR/Cas9 to generate a DGK theta endogenous promoter luciferase reporter HepG2 cell line, in which the endogenous DGK theta promoter controls the expression of the luciferase reporter gene. To test the cell line, FXR, the transcription factor for upregulating the expression of DGK theta gene, was used to validate the cell line. Furthermore, the reported agonists for the expression of DGK theta, cAMP and GW4064, the known inhibitor for DGK theta enzyme activity, R59949, and a potential regulator for DGK theta enzyme expression, EGCG (the major catechin in green tea), were applied to the reporter cell line. The results indicated that these reagents could significantly regulate the expression of reporter luciferase. Finally, four transcription factors (E2F1, c-Myc, USF1, and Bmal1) potentially binding to the DGK theta gene's upstream promoter region were tested. DGK theta expression was upregulated by c-Myc and downregulated by E2F1, which was also confirmed in wild-type HepG2 cells. We found that the cell line's luciferase activity was directly correlated with DGK theta endogenous promoter activity, suggesting that it is liable and sensitive for studying DGK theta transcriptional regulation. The study provides a useful tool for high-throughput drug screening for the treatment of DGK theta-involved diseases.