Melaleuca alternifolia concentrate (MAC) is the refined essential oil of the Australian native plant Melaleuca alternifolia. MAC has been reported to suppress the production of pro-inflammatory cytokines in both murine RAW264.7 macrophages and human monocytes stimulated with lipopolysaccharide (LPS). However, the mechanisms involved in this effect remain unclear. This study aims to delineate the molecular mechanisms that drive the anti-inflammatory activity of MAC and its active component, terpinen-4-ol, in macrophages. The effects of MAC on RAW264.7 cells were studied using western blotting, real-time PCR, an electrophoretic mobility shift assay (EMSA), and NF-κB luciferase reporter assays. Our results showed that MAC significantly increased both the mRNA and protein levels of heme oxygenase-1 (HO-1) via p38 and JNK MAPK activation. In addition, we showed that MAC significantly increased the activation and nuclear translocation of NF-E2-related factor 2 (Nrf2), a key transcription factor regulating HO-1 induction. MAC was also associated with significant inhibition of iNOS expression, NO production, and NF-κB activation. HO-1 was required for these anti-inflammatory effects as tin protoporphyrin IX (SnPPIX), an HO-1 inhibitor, abolished the effects of MAC on LPS-induced iNOS, NO, and NF-κB activation. Our results indicate that MAC induces HO-1 expression in murine macrophages via the p38 MAPK and JNK pathways and that this induction is required for its anti-inflammatory activity.