L-glutamine (Gln) is a nonessential amino acid that is the most abundant amino acid in plasma. Gln has been reported to have an anti-inflammatory activity that involves deactivation of mitogen-activated protein kinases (MAPKs) in a MAPK phosphatase (MKP)-1-dependent manner. This study investigated the role of Gln in the inhibition of DNFB-induced allergic contact dermatitis (CD) in the ears of mice, and specifically the involvement of Gln in p38 MAPK inhibition. Topical application of Gln or the p38 inhibitor, SB202190, suppressed DNFB-induced CD. Gln application inhibited DNFB-induced p38 phosphorylation. Western blot analysis revealed that Gln application resulted in early phosphorylation and protein induction of MKP-1. MKP-1 small interfering RNA (siRNA), but not control siRNA, abrogated Gln-mediated early phosphorylation, protein induction of MKP-1, deactivation of p38, and Gln-mediated suppression of CD. The extracellular signal-regulated kinase (ERK) inhibitor, U0126, blocked Gln-induced MKP-1 phosphorylation and protein induction, as well as Gln suppression of CD. These results suggest that Gln suppresses DNFB-induced CD via deactivation of p38 MAPK through the early induction of MKP-1, the negative regulator of p38, in an ERK-dependent manner.