Zanthoxylum myriacanthum var. pubescens (Maqian) is an economically important plant that is widely consumed as a flavoring agent or indigenous medicine by local ethnic groups in Southwest China. The pharmacological activities, such as the anti-inflammatory activity of Maqian essential oil, have been highlighted in recent years. In this study, the in vitro and in vivo anti-influenza activities of Maqian essential oil and its major ingredient, D/Llimonene, were assessed. The Maqian essential oil and D/L-limonene were effective against the influenza A and influenza B viruses in vitro. Notably, the Maqian essential oil was highly inhibitive against the drug-resistant influenza virus strain with an EC50 of 1.87 +/- 1.04 mu g/ml and a selective index of 85.13. Mode-of-action analysis revealed that Maqian essential oil and D/L-limonene inhibited the influenza virus at the stage of intracellular replication. For in vivo therapeutic efficacy, both intragastric and intranasal administration of D/L-limonene protected over 50% of mice from lethal influenza virus challenge. Both infectious viral particles and viral genome RNA were decreased approximately 2-fold in the D/L-limonene-treated mice compared with the virus group. Treatment with D/L-limonene potently improved the pathological features of the influenza virus-infected mice. Analysis of the gene expression level revealed that the major proinflammatory cytokines, including IL-1 beta, IL-6 and TNF-alpha, were significantly decreased in the D/L-limonene treated mouse lung tissues. Interestingly, we found that D-limonene was more potent than L-limonene in inhibiting proinflammatory cytokines. Transcriptome analysis revealed 32 host genes that participated in the antiviral process, most of which were related to the immune system and signal transduction. Combined with the antiviral and immunoregulatory effects of limonene, we believe that this multi-functional monoterpene has potential to become a novel candidate against influenza virus infections.