Tight junctions play an important role in maintaining barrier integrity of intestinal epithelia. Activation of AMP-activated protein kinase (AMPK) promotes tight junction assembly in intestinal epithelial cells (IEC). Fructo-oligosaccharides (FOS), well-known prebiotics, have previously been shown to alleviate inflammation-associated intestinal epithelial disruption although the mechanisms were unclear. This study aimed to investigate any effect of FOS on AMPK activity and tight junction assembly under non-inflammatory and inflammatory conditions using T84 cells as an IEC model. As analyzed by western blot, FOS induced AMPK activation through a calcium sensing receptor (CaSR)-phospholipase C (PLC)- Ca²⁺/calmodulin-dependent protein kinase kinase-β (CaMKKβ) pathway. Calcium switch assays and immunofluorescence staining of zonula occludens-1 (ZO-1) revealed that FOS induced tight junction assembly via an CaMKKβ-AMPK-dependent mechanism in IEC. Interestingly, FOS reversed the suppressive effect of lipopolysaccharide (LPS) on AMPK activity and tight junction assembly via a CaMKKβ pathway. Taken together, these findings uncover a prebiotic-independent effect of FOS in promoting intestinal epithelial tight junction assembly through AMPK activation, which may have implications for the treatment of diseases whose pathogenesis involves impaired intestinal barrier function.