The high-fat-diet-induced metabolic disorders are often accompanied with oxidative stress and inflammatory responses, which eventually results in lipotoxicity and tissue impairment. It is reported that probiotics have abilities to improve lipid metabolism and attenuate hyperlipidemia. In this study, all ICR mice were randomly divided into three groups, including a normal control group, high-fat-diet (HFD) group, and HFD + Lactobacillus acidophilus NX2-6 (LHLA) group (n = 8). Our results showed that 8-week oral administration (10⁹ cfu/mL) of L. acidophilus NX2-6 (NX2-6) improved the serum lipid profile in ICR mice using biochemical analysis. The Western blot method showed that NX2-6 treatment suppressed lipid accumulation in the adipose tissue by suppressing de novo lipogenesis and adipogenesis but promoting lipolysis and energy expenditure. However, supplementation with NX2-6 could not effectively ameliorate HFD-induced ER stress and inflammation in the adipose tissue. Meanwhile, NX2-6 also enhanced antioxidant capacity, improved the intestinal physical barrier and mitophagy, and suppressed inflammatory responses in the colon. High-throughput sequencing technology analysis showed that NX2-6 increased microbial species diversity and the relative abundance of beneficial bacteria, including Bacteroidetes, Verrucomicrobia, S24-7, Lactobacillaceae, Lactobacillus, and Parabacteroides but decreased the proportion of several harmful microorganisms, such as Proteobacteria, Desulfovibrionaceae, Alistipes, Desulfovibrio, Helicobacter, and Mucispirillum. Our data provide a new dietary insight into the prevention of diet-induced obesity. Specifically, this manuscript is presented on the anti-obesity effects of NX2-6 and underlying molecular mechanisms, which will provide a theoretical basis of anti-obesity probiotics for the development of functional foods.