Docosahexaenoic acid (DHA) has been reported to have protective effects against pulmonary hypertension (PH). The protective effect could be partially attributed to the direct effect on pulmonary artery smooth muscle cells (PASMCs). In the present study, we investigated the effect of DHA on interleukin-1β (IL-1β)-induced cyclooxygenase-2 (COX-2) expression in cultured human PASMCs. Cells were treated with DHA (30 μM) in the presence or absence of IL-1β (3 ng/ml). Atmospheric pressure of 60 mmHg which simulates severe PH was given to PASMCs. COX-2 protein expression transiently induced by IL-1β stimulation, peaking at 6–24 h and returning toward basal levels after 48 h. DHA did not affect the COX-2 protein expression after 24 h of IL-1β stimulation, but significantly prevented a decrease in the COX-2 protein expression at 48 h. This preventing effect of DHA was also observed in the pressurized cells simulating PH. DHA significantly increased a p38 mitogen-activated protein kinase (MAPK) phosphorylation at 48 h in both non-pressurized and pressurized cells. These results suggest that DHA enhances IL-1β-induced COX-2 expression time by activating of p38 MAPK in the delayed phase in PASMCs. The present study may contribute to the understanding of basic mechanisms underlying the beneficial effects of DHA on PH.