The rising pandemic of obesity has received significant attention. Yet, more safe and effective targeted strategies must be used to mitigate its impact on individual health and the global disease burden. While the health benefits of resistant starch (RS) are well-documented, the role of RT-90 (a phosphate-modified tapioca RS containing 90.1 % total dietary fiber) in mitigating obesity remains unknown. Accordingly, the physicochemical characteristics and protective effects of RT-90 on obesity were investigated in high-fat diet (HFD)-fed mice. Physicochemical property examinations showed that RT-90 consisted of small, round starch granules (D90: 20.69 ± 0.4 μm) with a crystalline structure, P-O-C stretching, and high peak melting temperature and enthalpy. Additionally, feeding mice with RT-90 significantly decreased body weight, improved oral glucose tolerance test (OGTT), reduced fatty liver and adipose tissue accumulation, lowered oxidative stress and inflammation by upregulating antioxidant enzymes (SOD, catalase, GPx) and anti-inflammatory cytokines (IL-4, IL-10), and enhanced fecal lipids and bile acid excretion. Notably, RT-90 administration in HFD-fed mice was associated with the inhibition of obesity-associated harmful bacteria ([Eubacterium]_xylanophilum group, Allobaculum, Clostridia_UCG-014, Dubosiella) and promotion of short-chain fatty acids (SCFAs)-producing gut flora, including Bifidobacterium, Lactobacillus, Bacteroides, and Parabacteroides, which suggests a possible mechanism by which RT-90 alters gut microbiota to attenuate obesity. These novel findings first revealed that RT-90 facilitates weight loss through its antioxidant, anti-inflammatory, and microbiota modulation abilities. This provides a promising strategy for managing overweight or obesity and holds potential for applications in developing healthy food products.