Background and aims <p>Microbial-mediated Zinc (Zn) biofortification, is an environmentally friendly strategy for sustainable agriculture. In this study, the effects of foliar spraying of endophytic bacteria from Zn hyperaccumulator <i>Sedum alfredii</i> Hance (<i>S. alfredii</i>) on the growth, Zn uptake and Zn nutritional quality of wheat (<i>Triticum aestivum</i> L.) were evaluated in a field experiment.</p> Methods <p>The isolated endophytic bacteria were cultivated into microbial fertilizers and applied at the jointing, heading, flowering and grain-filling stages of wheat. After harvest, the agronomic traits, Zn and other mineral element contents of wheat, as well as the contents of Zn and available Zn in the soil were determined. The physiologically-based extraction test (PBET) was used to evaluate the absorption of edible Zn in grains by the human body.</p> Results <p>Spraying endophytic bacteria optimized the wheat agronomic traits, increased the yield of wheat, and improved available Zn concentration in rhizosphere soil. Among the 10 strains, SaPA1, SaVA1, SaPS1, SaPS3, and SaEN1 showed notable abilities to promote the uptake and accumulation of Zn in wheat, with an increase rate of grain Zn concentration by up to 42.56%. Furthermore, those strains could not only reduce the phytic acid (PA) content but also increased the bioaccessible Zn concentration in grains. In addition, the concentration of other trace elements was also improved after sprayed certain strains.</p> Conclusion <p>Our research indicated that selected endophytic bacteria of Zn hyperaccumulator can be used as microbial fertilizers for biofortification and enhancing the nutrition of trace elements in crop.</p> Graphical Abstract <p></p>