Columbianetin acetate (CE) is one of the effective components of Angelica pubescens. So far, the specific role and molecular mechanism of CE in pancreatic cancer are not clear. Thus, this study aimed to explore the specific mechanism of CE on pancreatic cancer. The target genes combined with CE were predicted through the PharmMapper database and the 3D molecular structure of CE. Then, the Cancer Genome Atlas (TCGA) and Cistrome data browser (DB) databases were used to screen Meiotic nuclear divisions 1 (MND1)-related genes, transcription factors, and transcription factor data sets, and the intersection of the above data sets. The "limma" package in the R and gene expression profiling interactive analysis (GEPIA) databases were used to analyze the correlation and survival difference between the target genes and MND1 to predict the degree of association between CE and MND1. Western blotting and RT-PCR experiments revealed the regulatory relationship among CE, E2F1, and MND1 at the cellular level. The specific effects of CE on pancreatic cancer cells were explored through CCK8, wound healing, migration, and flow cycle experiments. E2F1, also the predictive transcription factor of MND1, was also the predictive target protein of CE. At the same time, E2F1 and MND1 were closely related in pancreatic tissue. In the cell function experiment, CE and interference with E2F1 expression could reduce the gene and protein expression of MND1, which was closely associated with cell proliferation, migration, and cycle development. Similarly, interfering with the expression of mnd1 can also inhibit the further development of tumor cells. CE may inhibit the development of pancreatic cancer cells by reducing the expression of MND1. This implies that CE may be a potential novel agent for the treatment of pancreatic cancer.