The solute carrier family 13 member 5 (SLC13A5) is a newly identified sodium-coupled transporter that mediates the cellular uptake of the tricarboxylic acid cycle (TCA) intermediate citrate, which plays an important role in the synthesis of fatty acid and energy homeostasis. Recently, the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), initially characterized as xenobiotic sensors, have been functionally linked to the regulation of various physiological processes that associated with lipid metabolism and energy homeostasis. Here, we show that SLC13A5 is robustly up-regulated by the prototypical CAR activator phenobarbital (PB) and PXR activator rifampicin (RIF) in human primary hepatocytes (HPHs). Two distal enhancer modules located upstream of the SLC13A5 gene transcription start site are identified and postulated as the key elements governing the induction of SLC13A5. In electrophoretic mobility shift assays, these elements demonstrated strong binding to PXR/CAR proteins in the presence of their heterodimer partner, retinoid X receptor. Cell-based reporter assays in HepG2 cells and HPHs further showed that SLC13A5 reporter construct, containing both elements could be transactivated by CAR and PXR upon exposure to PB and RIF, respectively. Moreover, our chromatin immunoprecipitation (CHIP) assays in HPHs showed that after PB and RIF exposure, significantly increased amounts CAR and PXR proteins were recruited to these distal enhancers. Together, our results clearly indicate that CAR and PXR are crucial for chemical-mediated induction of the SLC13A5 gene, and the regulation network of CAR/PXR-SLC13A5 may contribute to the overall energy homeostasis under metabolic and chemical stresses.