Nonalcoholic fatty liver disease (NAFLD) is the main cause of chronic liver disease worldwide. Previous evidence indicates that probiotics can be applied as a therapeutic agent for NAFLD. In this study, the potential probiotic strain Lactobacillus paracasei Jlus66 was isolated from natural fermented milk by a culture-dependent method, and its probiotic potentials were tested by established in vitro tests. In addition, the protective effect of Lactobacillus paracasei Jlus66 against NAFLD was evaluated in rat models. Compared with the high-fat-diet (HFD) group, the rats administered with 4 × 1010 cfu Jlus66 had significantly lower body weight gain, serum triglyceride (TG), low-density lipoprotein (LDL) as well as aminotransferase (ALT). Histopathological analysis showed Jlus66 also reduced the level of hepatic triglycerides and steatosis. From the above we conclude that L. paracasei Jlus66 has great potential as a probiotic in protecting from NAFLD.