The present study is the first one to investigate the glucosinolates (GLS) profile and anti-fibrotic effect of isothiocyanates (ITCs) rich fraction of Matthiola arabica (Brassicaceae) using an experimental model of liver fibrosis in rats. Five GLS (ethyl glucosinolate, gluconapin, glucodehydroerucin, glucoerucin and glucoraphanin) were identified by gas liquid chromatography-mass spectrometric (GLC-MS) analysis of their hydrolysis products, produced by the natural autolysis and exogenous myrosinase hydrolysis using one and two units of the enzyme. Spectrophotometric determination of the total intact GLS revealed that content in the fresh sample was 1.8 times higher than in the dry one. ITCs rich fraction was prepared by natural autolysis of the fresh aerial part. Male albino rats were given carbon tetrachloride (CCl4) (0.5 ml/kg, twice a week) and/or ITCs -rich fraction (30 mg/ kg, three times a week) for six weeks. Liver function, different oxidative stress, inflammatory and fibrosis markers were investigated. Treatment of animals with ITCs rich fraction significantly counteracted the changes in liver function induced by CCl4. Histopathological examination under both light and electron microscope showed the anti-fibrotic effect of ITCs rich fraction. This finding was confirmed with the markedly improved liver fibrosis markers with ITCs rich fraction co-treatment. In elucidation of anti-fibrotic mechanisms of ITCs rich fraction, the significant glutathione depletion and lipid peroxidation caused by CCl4 intoxication was restored by ITCs rich fraction co-treatment. Besides, ITCs rich fraction showed an anti-inflammatory effect through its ability to counteract the significant increase in nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2) expression, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels in liver tissue that caused by CCl4 intoxication. These findings indicate that ITCs-rich fraction of M. arabica possesses a promising anti-fibrotic effect which can be attributed to its antioxidant and anti-inflammatory properties.