Branched-chain fatty acids (BCFA) are generating interest for their benefits on human health particularly for their prebiotic effect on newborn gastrointestinal tract. Those fatty acids are synthesized by ruminal bacteria and incorporated into milk, the latter being the main source of BCFA in the North American diet. Some Bacillus genus species are used in animal production as direct-fed microbials and are known to synthesize BCFA to form their cell walls. Those probiotics have been shown to increase milk production and composition in small and large ruminants. To confirm that probiotics from the Bacillus genus can increase cow performance and affect milk fatty acid composition, 6 multiparous cows fitted with rumen cannula were used in a randomized replicated crossover design. Cows either received 200 g/d of whey powder as a control or 200 g/d of Bioplus 2B (Chr. Hansen, Milwaukee, WI), a commercial direct-fed microbial of Bacillus subtilis and Bacillus licheniformis, representing a daily dose of 6.4 × 10¹¹ cfu using whey powder as carrier. The 2 experimental periods lasted 14 d and a 7-d washout period was observed after each one. Samples were collected at d 0, 13 and 14 of each period. Milk production, composition and fatty acid profile as well as ruminal parameters and microbiota were evaluated. Data from d 0 were used as covariate. Treatments did not affect productivity and milk composition. The use of Bioplus 2B increased the relative concentration of anteiso 13:0 and anteiso 15:0 (P < 0.05) and tended to increase total concentration of BCFA (P < 0.10) compared with control. Treatments did not affect ruminal pH, ammonia nitrogen and concentrations of acetate, propionate and butyrate. However, Bioplus 2B increased concentration of isovalerate (P < 0.05) and tended to increase the concentration of isobutyrate (P < 0.10). This trial indicates that milk fatty acid profile is sensitive to ruminal microbiota modifications. Probiotics of the Bacillus genus could be used as part of a BCFA enhancing protocol to increased those fatty acids in dairy products.