Odontoblasts are derived from dental papilla mesenchymal cells and have an important role in defense against bacterial infection, whereas autophagy can recycle long-lived proteins and damaged organelles to sustain cellular homeostasis. Thus, this study explores the role of autophagy in odontoblast differentiation with lipopolysaccharide (LPS) stimulation in vitro and the colocalization of p-NF-κB and LC3 in caries teeth. The odontoblasts differentiation was enhanced through LPS stimulation, and this outcome was reflected in the increased number of mineralized nodules and alkaline phosphatase (ALP) activity. The expression levels of the autophagy markers LC3, Atg5, Beclin1 and TFE3 increased time dependently, as well along with the amount of autophagosomes and autophagy fluxes. This result suggests that autophagy was enhanced in odontoblasts cultured with mineralized-induced media containing LPS. To confirm the role of autophagy in differentiated odontoblasts with LPS stimulation, chloroquine (CQ) or rapamycin were used to either block or enhance autophagy. The number of mineralized nodules decreased when autophagy was inhibited, but this number increased with rapamycin treatment. Phosphorylated nuclear factor-κB (NF-κB) expression was negatively related to autophagy and could inhibit odontoblast differentiation. Furthermore, p-NF-κB and LC3 colocalization could be detected in cells stimulated with LPS. The nucleus translocation of p-NF-κB in odontoblasts was enhanced when autophagy was inhibited by Atg5 small interfering RNA. In addition, the colocalization of p-NF-κB and LC3 in odontoblasts and sub-odontoblastic layers was observed in caries teeth with reactionary dentin. Therefore, our findings provide a novel insight into the role of autophagy in regulating odontoblast differentiation by suppressing NF-κB activation in inflammatory environments.