AbstractThis study aimed to determine the efficacy of a bacterial 6-phytase (Buttiauxella spp.) supplemented beyond traditional dose levels based on jejunal mucosa-associated microbiota, apparent ileal digestibility (AID), intestinal health and bone parameters, and growth performance of nursery pigs. Seventy-two weaned pigs (36 barrows and 36 gilts at 21 d of age with 5.8 ± 0.5 kg BW) were allotted to six treatments based on randomized complete block design with sex and initial BW as blocks and fed in three dietary phases (phase 1 for 14 d, phase 2 for 10 d, and phase 3 for 14 d). The treatments included a negative control (NC) diet without phytase formulated meeting nutrient requirements by NRC and the other five treatments were deficient in calcium (Ca) and phosphorus (P) by 0.12% with increasing levels of a bacterial 6-phytase (0, 500, 1,000, 2,000, and 5,000 FTU/kg feed). Titanium dioxide (0.4%) was added to phase 3 diets as an indigestible marker to measure AID of nutrients. On day 45, all pigs were euthanized to collect ileal digesta to measure AID, the third metacarpus to measure bone parameters, and jejunal mucosa to evaluate intestinal health and microbiota. Data were analyzed using the MIXED procedure for polynomial contrasts and the NLMIXED procedure for broken line analysis using the SAS 9.4. Broken line analysis demonstrated that 948 FTU/kg feed increased (P< 0.05) the ADG and the bone P content. Increasing phytase supplementation increased (linear,P< 0.05) AID of CP, bone P, and ash content. Increasing phytase supplementation reduced (P< 0.05) the fecal score during phases 2 and 3. Broken line analysis demonstrated that 1,889 FTU/kg feed increased (P< 0.05) bone breaking strength. Increasing phytase supplementation (PC vs. Phy) increased (P< 0.05) AID of ether extract (EE) and P. The supplementation of phytase at 2,000 FTU/kg feed tended (P= 0.087) to reduce the relative abundance ofPrevotellaceae. In conclusion, the supplementation of a bacterial 6-phytase beyond traditional dose levels improved bone breaking strength, bone ash, and P content, AID of CP, EE, and P, and growth performance of nursery pigs with reduced relative abundance of Bacteroidetes specificallyPrevotellaceaein the jejunal mucosa. Supplementation of a bacterial 6-phytase between 1,000 and 2,000 FTU/kg feed provided benefits associated with growth performance and bone parameters of nursery pigs.