Pancreatic cancer (PC) is becoming one of the deadliest cancers, with mortality among the highest worldwide because of its pathogenic latency and the lack of efficient drugs in the clinic. Considering that cancer cells undergo proliferation and differentiation at substantial metabolic costs, as indicated by dysregulated glycolysis and an abnormal TCA cycle induced by mitochondrial damage, we investigated the therapeutic capacity of berberine (BBR) in pancreatic cancer using a cell metabolomics method. A phenotypic assay revealed the significant inhibitory role of BBR in PC cell viability and metastasis. In addition, a precision-targeted metabolome assay showed that BBR profoundly dysregulated the energy metabolism of PC cells, and phenotypic observations based on imaging indicated that PC cell mitochondria were markedly damaged after BBR treatment. Notably, citrate metabolism and transportation in cell mitochondria were significantly influenced by BBR, which led to the blocked biosynthesis of the defined fatty acids (FAs) through the regulation of ACLY, ACO1, and SLC25A1. Therefore, the regulatory effects of FAs on PC cell proliferation and metastasis may be regulated by BBR through targeting citrate metabolism. Collectively, our in vitro data preliminarily reveals the therapeutic potential of BBR against pancreatic cancer by targeting citrate metabolism, citrate might be a new target for drug development and the treatment against PC, but further experimental verification will be required subsequently. Moreover, our study demonstrated that the cell metabolomics method pertains to the capacity to rapidly explore biochemical functions of natural products.