The influence of sodium reduction and flavor enhancer addition of probiotic Prato cheese (L. casei 01, 6 log cfu/g mL) on fatty acid profile during 1 (after the manufacturing), 30 (ripening) and 60 (commercial shelf life) days was investigated. Five formulations of probiotic Prato cheese were prepared: CI (control - 100% NaCl), CII (1 NaCl:1 KCl (wt/wt)), CIII (1 NaCl:1 KCl (wt/wt) + 1% wt/wt arginine), CIV (1 NaCl:1 KCl (wt/wt) + 1% wt/wt yeast extract), CV (1 NaCl:1 KCl (wt/wt) +1% wt/wt oregano extract). Overall, no change was observed on the fatty acid profile among cheeses (P < 0.05); however, slight changes along the storage time (P > 0.05) were observed. Among the saturated fatty acids, the reduced sodium probiotic Prato cheeses showed higher levels of myristic, palmitic and stearic acid (values ranged from 8.24 to 10.90; 22.88 to 27.85 and 12.62 to 14.75 g/100g fat, respectively, P > 0.05) without effect) when compared with the control cheese. In addition, Prato cheeses were characterized by higher levels of monounsaturated fatty acids (MUFAs), especially oleic acid (values ranged from 28.28 to 35.870, CIII and CII, respectively P > 0.05). Along the storage time it was observed a high decrease in short-chain fatty acids (SCFA) (values decreased from 10 to 3 g/100g fat, P > 0.05) and a subtle decrease in medium chain fatty acids (MCFA) (values decreased from 9 to 8 g/100g fat, P > 0.05). On the reverse, an increase in Long chain fatty acids (LCFA) levels was also noted for all cheeses (values ranged from 77.19 to 88.89 g/100g fat, CII and CV, respectively, P > 0.05). Overall, the findings report the effect of ripening and storage time contributed to some differences in fatty acid profile of reduced-sodium probiotic Prato cheeses.