Inhibitor of DNA binding 1 (ID1) has an aberrantly high expression in multiple cancer tissues, including colon cancer, lung cancer, breast cancer, and so on, which is closely related to cancer aggressiveness and poor clinical outcomes in cancer patients. It has been reported that ID1 maintains colorectal cancer cells (CRCs) stemness traits and contributes to the CRC drug resistance. While, the biological molecular mechanisms have not been fully elucidated. In this research, we found that ID1 upregulates octamer binding transcription factor (OCT4) protein level as well as OCT4 signaling pathway via Western blot, gene set enrichment analysis (GSEA), dual-luciferase reporter assay, and real-time PCR. Through the in vitro sphere formation assay, we found that overexpression of OCT4 reverses the inhibitory effect of knocking down ID1 on CRC sphere formation ability. With the help of JASPAR and GEPIA database, we predicted a novel transcriptional repressor-forkhead box D3 (FOXD3) of OCT4. Finally, by using co-immunoprecipitation (Co-IP), confocal and real-time PCR, we demonstrated that ID1 interacts with FOXD3 to inhibit its transcriptional repression activity and therefore to upregulate OCT4 transcription and OCT4 signaling pathway. In conclusion, this study provides a new theoretical basis for the regulation mechanism of colon cancer stem cells, and the newly found protein-protein interaction of ID1-FOXD3 provides a potential drug target for the therapy of CRC.