Systemic polyunsaturated fatty acids (PUFAs) were shown to improve the symptoms of dry eye syndrome due to their anti-inflammatory effects. This study evaluated the in vitro anti-inflammatory effects of PUFAs on human corneal epithelial (HCE) cells. HCE cells were incubated for 2 hours with different concentrations of PUFAs: alpha-linolenic acid (ALA), gamma-linolenic acid (GLA), and linoleic acid (LA). Oleic acid (OA) and dexamethasone (DM) served as negative and positive controls, respectively. Cells were stimulated with either polyinosinic:polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) complex. The protein contents and mRNA expression levels of IL-6, IL-8, IL-1β, and TNF-α were evaluated with multiplex fluorescent bead immunoassay and real-time PCR, respectively. The expression of inhibitory factor-κBα (I-κBα) was evaluated with real-time PCR. The protein and mRNA levels of IL-6, IL-8, IL-1β, and TNF-α were significantly increased after stimulation with LPS or poly I:C. Following treatment with ALA, a significant decrease was demonstrated in the protein content of TNF-α to 23.81% (P < 0.001), IL-6 to 46.71% (P < 0.001), IL-1β to 20.86% (P < 0.05), and IL-8 to 52.21% (P < 0.001). Similar results were demonstrated at the mRNA level. The anti-inflammatory effects of ALA were similar to those of DM for all of the pro-inflammatory cytokines. The ALA inhibition of the pro-inflammatory cytokines was associated with a significant reduction of I-κBα. ALA may serve as a potent anti-inflammatory agent in ocular surface inflammation. The anti-inflammatory effects of ALA are comparable to those of corticosteroids, and are mediated through NF-κB signal transduction.