The anti-inflammatory effect of sea cucumber peptides (SCP) in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages was tested. SCP significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression without affecting the cell viability. The mRNA expression of LPS-induced inflammatory cytokines including tumour necrosis factor-α, interleukin (IL)-1β and IL-6 was suppressed. SCP inhibited LPS-induced degradation of the inhibitor of κBα (IκBα) and nuclear transposition of NF-κB p65, resulting in decreased NF-κB transactivation. Moreover, SCP suppressed the LPS-induced phosphorylation of JNK, ERK and p38. In addition, the expression of heme oxygenase (HO)-1 in macrophages was up-regulated by SCP in a dose-dependent manner. The inhibition effect of SCP on the mRNA expression of LPS-induced inflammatory cytokines was partially reversed by co-treatment with a HO-1 inhibitor. The SCP with anti-inflammatory activity was made up of low-molecular-weight peptides rich in glycine, glutamic acid and aspartic acid. Collectively, these results demonstrate that SCP exerts anti-inflammatory function through inhibiting NF-κB and MAPK activation and inducing HO-1 expression in macrophages.