Due to their widespread use as model systems and their reputation as living fossils, horseshoe crabs (Xiphosura) have been studied intensively by physiologists and paleontologists. The close phylogenetic relationship between horseshoe crabs and arachnids might also have been expected to inspire studies of xiphosurans by comparative arachnologists, but surprisingly few have been undertaken. Here, the first exhaustive survey of muscular anatomy of the Atlantic horseshoe crab is conducted as part of an on-going study of the evolutionary morphology and phylogeny of arachnids. Dissections of adult and immature individuals established 113 muscle groups comprising over 750 individual muscles, with several being recognized or correctly described for the first time. New insights into skeletomuscular evolution and phylogeny of arachnids were derived primarily from the axial muscle system. Specifically, it is argued that Limulus retains a box-truss axial muscle system like that of plesiomorphic members of other arthropod groups, that this is also a plesiomorphic condition for Chelicerata, and that arachnids are united by the loss of one component of this system, the anterior oblique muscles. Combined with comparative morphological and molecular evidence from previous studies, this study adds greater weight to the widely held view that, among extant chelicerates, Xiphosura and Arachnida are monophyletic sister groups and counters recent speculation that scorpions are more closely related to xiphosurans than to spiders, whipscorpions and other arachnids.