Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic dysfunction for which effective interventions are lacking. To investigate the effects of resistant starch (RS) as a microbiota-directed dietary supplement for NAFLD treatment, we coupled a 4-month randomized placebo-controlled clinical trial in individuals with NAFLD (ChiCTR-IOR-15007519) with metagenomics and metabolomics analysis. Relative to the control (n = 97), the RS intervention (n = 99) resulted in a 9.08% absolute reduction of intrahepatic triglyceride content (IHTC), which was 5.89% after adjusting for weight loss. Serum branched-chain amino acids (BCAAs) and gut microbial species, in particular Bacteroides stercoris, significantly correlated with IHTC and liver enzymes and were reduced by RS. Multi-omics integrative analyses revealed the interplay among gut microbiota changes, BCAA availability, and hepatic steatosis, with causality supported by fecal microbiota transplantation and monocolonization in mice. Thus, RS dietary supplementation might be a strategy for managing NAFLD by altering gut microbiota composition and functionality.