Members of the phylum Apicomplexa actively enter host cells by a process involving the discharge of the apically localized microneme and rhoptry organelles. To unravel the processes involved in rhoptry organelle biogenesis, we focused on the Toxoplasma gondii armadillo repeats only protein (TgARO), a conserved acylated protein homogenously anchored to the rhoptry membrane. Conditional disruption of TgARO results in the random cytosolic dispersion of rhoptries and a severe defect in T. gondii invasion, with no effects on intracellular growth or host cell egress. Importantly, rhoptry displacement upon ARO depletion can be functionally complemented with wild-type TgARO but not an acylation mutant. TgARO interacts with myosin F, and inhibition of actin polymerization or myosin function also results in rhoptry dispersal, indicating that the apical positioning of rhoptries is an actomyosin-based process. Thus, TgARO mediates the apical localization of rhoptries, which is specifically required for host cell invasion.