Little is known about the reciprocal effect of manganese-oxidizing bacteria (MOB), in situ-generated biogenic manganese oxide (BMO) and thallium (Tl) removal. In this study, the Tl(I) removal characteristics during extracellular BMO formation by Pseudomonas putida strain MnB1 were investigated. BMO with a final maturity of 91.25 % achieved 95.20 % Tl(I) removal during biogenic Mn(II) oxidation. The improvement mechanisms of Tl(I) removal in biological systems were mainly due to the involvement of Mn(III) intermediates and tyrosine and tryptophan protein-like substances from MOB. The addition of exogenous MOB reoxidized the residual Mn²⁺ to BMO, thus promoting Tl(I) removal. BMO showed a higher adsorption ability for Tl(I) at inner depths owing to its layer stacking structure, and 64.02 % Tl(I) was further oxidized to Tl(III) by highly matured BMO. Surface complexation also played an important role during Tl(I) removal. Moreover, BMO showed good adsorption selectivity for Tl(I) with coexisting Fe(III), Cr(III) and Cd(II). These findings expand existing knowledge on the characteristics of in situ-generated BMO and indicate that BMO has practical application value in Tl(I) decontamination from water.