The aim of this study was to determine effect of diet-based seasonal changes on fatty acid composition of Jersey milk in mountain region of Croatia. Bulk milk samples (200 mL) were collected during total-mixed ratio (TMR)-based, intermediate and pasture-based diet. Fatty acid composition of milk was determined by gas-chromatography. We found that pasture-based diet has positive effect on fatty acid composition of Jersey milk considering human nutrition. During pasture-based diet, Jersey cows produced milk with lower (P<0.05) saturated fatty acid and higher (P<0.05) polyunsaturated fatty acid content. Namely, regarding individual fatty acids, milk produced during pasture-based diet had lower (P<0.05) C16:0 and higher (P<0.05) C18:2n-6, C18:3n-6 and conjugated linoleic acid (CLA) content than milk produced during TMR and intermediate diet. Milk produced during pasture-based diet can be valuable source of health-beneficial PUFA in diet.