The nucleotide binding oligomerization domain containing 2 (NOD2) protein and its ligand N-acetyl muramyl dipeptide (MDP) are crucially involved in Crohn's disease (CD). However, the mechanism by which NOD2 signaling is regulated in CD patients remains unclear. Ubiquitin specific protease (USP14) is a deubiquitylase that plays an important role in immunity. This study aimed to investigate the mechanism by which UPS14 regulates NOD2 induced inflammatory response in CD and inflammatory bowel diseases (IBD). Our results showed that USP14 protein and mRNA levels in intestinal tissues of CD patients were significantly higher than those in healthy controls. In addition, USP14 was upregulated in IBD mouse model. While treatment with MDP, TNF-α or the Toll-like receptor 1/2 agonist Pam3CSK4 all led to significantly higher mRNA levels of TNF-α, IL-8 and IL-1β in THP-1 cells, pretreatment with USP14 inhibitor IU1 could stimulate further upregulation of TNF-α, IL-8 and IL-1β. In particular, MDP promoted the activation of JNK, ERK1/2 and p38 as well as NF-kB in THP-1 cells, and IU1 significantly enhanced the MDP-induced activation of these proteins without effects on USP14 protein level. Furthermore, the JNK inhibitor sp600125, ERK1/2 inhibitor U0126 or P38 MAPK inhibitor PD169316 significantly decreased the mRNA levels of TNF-α, IL-8 and IL-1β in THP-1 cells stimulated by both IU1 and MDP. In conclusion, our findings suggest that USP14 could inhibit MDP-induced activation of MAPK signaling and the inflammation response involved in IBD, and that USP14 is a potential therapeutic target for IBD.