Multiple molecular cues guide neuronal axons to their targets during development. Previous studiesin vitrohave shown that mechanical stimulation also can affect axon growth; however, whether mechanical force contributes to axon guidancein vivois unknown. We investigated the role of muscle contractions in the guidance of zebrafish peripheral Rohon–Beard (RB) sensory axonsin vivo. We analyzed several mutants that affect muscle contraction through different molecular pathways, including a new mutant allele of thetitin a (pik)gene, mutants that affect the hedgehog signaling pathway, and a nicotinic acetylcholine receptor mutant. We found RB axon defects in these mutants, the severity of which appeared to correlate with the extent of muscle contraction loss. These axons extend between the muscle and skin and normally have ventral trajectories and repel each other on contact. RB peripheral axons in muscle mutants extend longitudinally instead of ventrally, and the axons fail to repel one another on contact. In addition, we showed that limiting muscle movements by embedding embryos in agarose caused similar defects in peripheral RB axon guidance. This work suggests that the mechanical forces generated by muscle contractions are necessary for proper sensory axon pathfindingin vivo.