How to effectively remove excess Sb(III) in the water environment by biosorption is receiving close attention in the international scientific community. To obtain the maximum biosorption efficiency, response surface methodology (RSM) was employed to optimize a total of 13 factors for biosorption of Sb(III) onto living Rhodotorula mucilaginosa DJHN070401. The mechanism of biosorption and bioaccumulation was also studied. The results showed that biosorption reached 56.83% under the optimum conditions. Besides, pH, Fe2+, and temperature are significant influencing factors, and control of Ca2+ and Fe2+ has a beneficial impact on Sb(III) biosorption. The characterization explained that physical adsorption occurred readily on the loose and porous surface of DJHN070401 where carboxyl, amidogen, phosphate group, and polysaccharide C-O functional groups facilitated absorption by complexation with Sb(III), accompanied by ion exchange of Na+, Ca2+ ions with Sb(III). It was also noted that the living cell not only improved the removal efficiency in the presence of metabolic inhibitors but also prevented intracellular Sb(III) being re-released into the environment. The results of this study underpin improved and efficient methodology for biosorption of Sb(III) from wastewater.