Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) have anti-inflammatory effects. One specific PUFA, alpha-linolenic acid (ALA), shows both anti-inflammatory and antioxidant properties. In the testes, inflammatory mediators are known to increase when orchitis is induced by bacterial lipopolysaccharide (LPS). This study aimed to determine whether the anti-inflammatory properties of ALA could have a protective effect against LPS-induced orchitis in mice. The mice were divided into untreated control, orchitis and ALA-treated orchitis groups. Orchitis was induced by intraperitoneal injection of LPS. The ALA-treated group was administered ALA by gavage three days before intraperitoneal LPS injection. Cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), inducible nitric oxide synthase (iNOS) enzymes and nuclear factor kappa-B (NF-kappa B) in the testes, as well as serum interleukin 6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha), were analysed using enzyme-linked immunosorbent assay (ELISA) tests. LPS administration increased the expression of several inflammatory mediators, including IL-6, TNF-alpha and NF-kappa B, as well as the COX-2, cPLA2 and iNOS enzymes. ALA administration significantly prevented the LPS-induced increases in these inflammatory mediators and enzymes (p < .05). The anti-inflammatory and antioxidant effects of ALA may make it a useful candidate for the treatment of orchitis caused by bacterial LPS.