The alphaproteobacterium has the intriguing ability to navigate within magnetic fields, a behavior named magnetotaxis, governed by the formation of magnetosomes, intracellular membrane-enveloped crystals of magnetite. Magnetosomes are aligned in chains along the cell's motility axis by a dedicated multipart cytoskeleton ("magnetoskeleton"); however, precise estimates of its significance for magnetotaxis have not been reported. Here, we estimated the alignment of strains deficient in various magnetoskeletal constituents by live-cell motility tracking within defined magnetic fields ranging from 50 μT (reflecting the geomagnetic field) up to 400 μT. Motility tracking revealed that Δ and Δ strains (which assemble mispositioned and fragmented chains, respectively) are partially impaired in magnetotaxis, with approximately equal contributions of both proteins. This impairment was reflected by a required magnetic field strength of 200 μT to achieve a similar degree of alignment as for the wild-type strain in a 50-μT magnetic field. In contrast, the Δ strain, which predominantly forms clusters of magnetosomes, was only weakly aligned under any of the tested field conditions and could barely be distinguished from a nonmagnetic mutant. Most findings were corroborated by a soft agar swimming assay to analyze magnetotaxis based on the degree of distortion of swim halos formed in magnetic fields. Motility tracking further revealed that swimming speeds of are highest within the field strength equaling the geomagnetic field. In conclusion, magnetic properties and intracellular positioning of magnetosomes by a dedicated magnetoskeleton are required and optimized for bacterial magnetotaxis and most efficient locomotion within the geomagnetic field. In , magnetosomes are aligned in quasi-linear chains in a helical cell by a complex cytoskeletal network, including the actin-like MamK and adapter MamJ for magnetosome chain concatenation and segregation and MamY to position magnetosome chains along the shortest cellular axis of motility. Magnetosome chain positioning is assumed to be required for efficient magnetic navigation; however, the significance and contribution of all key constituents have not been quantified within defined and weak magnetic fields reflecting the geomagnetic field. Employing two different motility-based methods to consider the flagellum-mediated propulsion of cells, we depict individual benefits of all magnetoskeletal constituents for magnetotaxis. Whereas lack of resulted almost in an inability to align cells in weak magnetic fields, an approximately 4-fold-increased magnetic field strength was required to compensate for the loss of or In summary, the magnetoskeleton and optimal positioning of magnetosome chains are required for efficient magnetotaxis.