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We sought to determine if 28 days of probiotic supplementation influenced the plasma amino acid (AA) response to acute whey protein feeding. METHODS: Twenty-two recreationally active men (<i>n</i> = 11; 24.3 ± 3.2 yrs; 89.3 ± 7.2 kg) and women (<i>n</i> = 11; 23.0 ± 2.8 yrs; 70.2 ± 15.2 kg) participated in this double-blind, placebo-controlled, randomized study. Before (PRE) and after 28 days of supplementation (POST), participants reported to the lab following a 10-hr fast and provided a resting blood draw (0 min), then subsequently consumed 25 g of whey protein. Blood samples were collected at 15-min intervals for 2 h post-consumption (15–120 min) and later analyzed for plasma leucine, branched-chain AA (BCAA), essential AA (EAA), and total AA (TAA). Participants received a probiotic (PROB) consisting of 1 x10-9 colony forming units (CFU) <i>Bacillus subtilis</i> DE111 (<i>n</i> = 11) or a maltodextrin placebo (PL) (<i>n</i> = 11) for 28 days. Plasma AA response and area under the curve (AUC) values were analyzed via repeated measures analysis of variance. RESULTS: Our analysis indicated no significant (<i>p</i> < 0.05) differential responses for plasma leucine, BCAA, EAA, or TAA between PROB and PL from PRE to POST. AUC analysis revealed no group × time interaction for plasma leucine (<i>p</i> = 0.524), BCAA (<i>p</i> = 0.345), EAA (<i>p</i> = 0.512), and TAA (<i>p</i> = 0.712). CONCLUSION: These data indicate that 28 days of <i>Bacillus subtilis</i> DE111 does not affect plasma AA appearance following acute whey protein ingestion.