To investigate the potential effect of endotoxin tolerance on nucleotide­-binding oligomerization domain containing 2 (NOD2) signaling pathway, murine macrophage RAW264.7 cells were pretreated with lipopolysaccharide (LPS, 100 ng/mL) or phosphate buffered saline (PBS) for 20 h to establish endotoxin tolerance group and control group respectively. Both groups were subjected to the treatment with LPS (1 000 ng/mL) or heat­killed Aspergillus fumigatus (HAf). The mRNA levels of NOD2, receptor-­interacting protein 2 (RIP2) and tumor necrosis factor α (TNF-­α) were measured at 0, 2, 6, 12, 24 h after stimulation by real-­time quantitative polymerase chain reaction (PCR). The concentrations of secreted TNF-­α and interleukin 8 (IL­-8) were also quantified by enzyme-­linked immunosorbent assay (ELISA). The collective results showed that neither LPS nor HAf had any significant effect on NOD2, RIP2, and TNF­-α transcription or TNF­-α and IL­-8 secretion in the endotoxin tolerance group. On the contrary, a clear up-­regulation was detected in the control group upon either LPS or HAf treatment, concomitant with a peak at 12 h after stimulation. These data indicate that the NOD2 signaling pathway might be suppressed under endotoxin tolerance conditions.