Starch is an important dietary carbohydrate ingredient in ruminants’ diets, and can influence rumen fermentation, microbiota, methanogenesis and fiber digestibility. Dietary starch can influence both ruminal pH and hydrogen (H2). The present study was designed to manipulate ruminal H2 and pH through increasing starch intake, and investigate association of pH and dissolved H2 (dH2) with microbiota composition and fiber digestibility by using forward stepwise multivariate linear regression analysis. Twenty growing beef bulls were allocated into two groups, which were fed either low (LS) or moderate starch (MS) diets. About 43.2 % increase in dietary starch content was achieved by replacing 10 % of napier grass silage in the LS diet with 10 % ground corn grain, leading to 40.7 % of increase in starch intake. We measured feed intake and digestibility, rumen dissolved gases and fermentation and the abundance of microbial populations. Comparing with the LS diet, cattle fed the MS diet had increased (P < 0.05) feed digestibility, dH2 and methane (dCH4) concentration, VFA concentration, 18S rRNA gene copies of protozoa, 16S rRNA gene copies of methanogens and Selenomonas ruminantium, and decreased (P < 0.05) NDF and ADF digestibility, ruminal pH and 16S rRNA gene copies of Fibrobacter succinogenes. Forward stepwise multivariate linear regression analysis showed that ruminal pH was the variable most associated with NDF (R2 = 0.48; P = 0.001) and ADF digestibility (R2 = 0.74; P < 0.001), and 16S rRNA genes copies of F. succinogenes (R2 = 0.60; P < 0.001), while ruminal dH2 was the variable most associated with dCH4 concentration (R2 = 0.64; P < 0.001). Ruminal pH and dH2 were positively correlated with fiber digestibility and dCH4 concentration, respectively. In summary, enhanced ruminal methanogenesis and decreased fiber digestibility induced by increasing starch intake are possibly mediated through mechanisms involving an increase in ruminal dH2 concentration and a decrease in ruminal pH, respectively.