To observe the effect of Safflor Yellow (SY) Injection on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. Seventy-two mice were divided into six groups: control (saline + saline); LPS (LPS + saline); SY Injection [LPS + SY (10, 20 or 40 mg/kg, intravenously)] and anisodamine (AD) (LPS + AD). Thirty minutes after SY or AD administration, 15 mg/kg LPS was given intraperitoneally. All animals were sacrificed 4 h after LPS injection. Arterial blood gas and lung water content index (LWCI) were measured. Lung tissue myeloperoxidase (MPO) activity was assayed. mRNA expression of inflammatory cytokines was assayed by reverse-transcription polymerase chain reaction. Lung morphological and nuclear factor (NF)-kappa B p65-positive cell changes were observed by HE and immunohistochemical staining. p38 mitogen-activated protein kinase (MAPK) phosphorylation was observed by Western blotting. After LPS administration, all animals displayed increased arterial carbon dioxide partial pressure (PaCO2) and decreased arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SO2), HCO3 (-) concentration and pH, and increased LWCI, MPO activity, interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha mRNA expression, NF-kappa B p65-positive staining and p38 MAPK activation compared with normal controls (all P < 0.01). SY Injection significantly mitigated the LPS-induced increase in arterial PaCO and the decreases in arterial PaO2, SO2 and pH, and attenuated increases in LWCI and lung tissue MPO activity (all P < 0.01). Moreover, SY Injection inhibited the increases in NF-kappa B p65 staining and in TNF-alpha, IL-1 beta and IL-6 mRNA expression (all P < 0.01), and promoted the expression of the antiinflammatory cytokine IL-10 (P < 0.05) following LPS injection. LPS-induced pulmonary p38 MAPK phosphorylation was suppressed by pretreatment with SY Injection (P < 0.01). SY Injection ameliorates inflammatory ALI induced by LPS in mice.