Citrus pulp is a highly abundant by-product of the citrus industry. The aim of this study was to assess the effects of replacing extruded maize (EM; 20% of total diet) by dried citrus pulp (DCP; 20%) in a mixed diet on rumen fermentation and microbial populations in Rusitec fermenters. The two diets contained 50% alfalfa hay and 50% concentrate, and the same protein level. Four Rusitec fermenters were used in a cross-over design with two 13-d incubation runs. After 7-d of diet adaptation, diet disappearance, fermentation parameters, microbial growth, and microbial populations were assessed. Fermenters receiving the DCP showed greater pH values and fiber disappearance (p < 0.001) and lower methane production (p = 0.03) than those fed EM. Replacing EM by DCP caused an increase in the proportions of propionate and butyrate (p < 0.001) and a decrease in acetate (p = 0.04). Microbial growth, bacterial diversity, and the quantity of bacteria and protozoa DNA were not affected by the diet, but the relative abundances of fungi and archaea were greater (p < 0.03) in solid and liquid phases of DCP fermenters, respectively. Results indicate that DCP can substitute EM, promoting a more efficient ruminal fermentation.