Background and Aim:Resistant starch (RS) is difficult to digest in the digestive tract. This study aimed to evaluate the effects of heat-moisture treatment (HMT) on RS in cassava and examined its impact on rumen fermentation.Materials and Methods:Cassava flour was used as a raw material and used in a randomized block design with four different cycles of HMT as the treatments and four different rumen incubationsin vitroas blocks. Treatments included: HMT0: without HMT (control), HMT1: one HMT cycle, HMT2: two HMT cycles, and HMT3: three HMT cycles. Heat-moisture treatment processes were performed at 121°C for 15 min and then freezing at −20°C for 6 h. Analyzed HMT cassava starch characteristics included components, digestibility, and physicochemical properties. Inin vitrorumen fermentation studies (48 h incubation) using HMT cassava, digestibility, gas production, methane, fermentation profiles, and microbial population assessments were performed.Results:Heat-moisture treatment significantly reduced (p < 0.05) starch, amylopectin, rapidly digestible starch (RDS), and slowly digestible starch levels. In contrast, amylose, reducing sugars, very RDS, RS, and protein digestion levels were significantly increased (p < 0.05). Additionally, a reduced crystallinity index and an increased amorphous index were observed in starch using Fourier-transform infrared analyses, while a change in crystalline type from type A to type B, along with a reduction in crystallinity degree, was observed in X-ray diffraction analyses. Heat-moisture treatment significantly (p < 0.05) reduced rumen dry matter (DM) degradation, gas production, methane (CH4for 12 h), volatile fatty acid (VFA), and propionate levels. In addition, acetate, butyrate, and acetate/propionate ratios, as well as population ofStreptococcusbovisandBacteroideswere significantly increased (p < 0.05). However, pH, ammonia, and organic matter digestibility were unaffected (p > 0.05) by HMT.Conclusion:Cassava HMT altered starch characteristics, significantly increased RS, which appeared to limit rumen digestion activity, decreased rumen DM degradation, gas production, VFAs, and CH4production for 12 h, but increasedS. bovisandBacteroideslevels.