Canine heartworm disease is caused by the filarial nematode Dirofilaria immitis, which has been found on all continents except Antarctica. The endosymbiont of several filarial nematode species, Wolbachia, is crucial in the reproduction, development, and survival of the filarial nematode. Wolbachia also contributes to immunomodulation in the vertebrate hosts of the nematodes, supporting the establishment of infection. The investigation of the characteristics of this endosymbiont provided novel insights into drug targets for filarial disease control and treatment. In the process of treating filarial nematodes, the acute or chronic release of Wolbachia can be concerning as Wolbachia induces inflammatory reactions, which lead to pathologic lesions. Doxycycline targets Wolbachia and has been utilized in the treatment of onchocerciasis and dirofilariasis. In heartworm treatment, the American Heartworm Society (AHS) recommends a doxycycline treatment at 10 mg/kg twice daily for 28 days before adulticidal treatment. However, the phenotypes and mechanisms of the impact of doxycycline on Wolbachia and, consequently, D. immitis, require further investigation. In this dissertation, chapter 2 discusses a possible mechanism behind the phenotype that doxycycline-treated microfilariae (mf) cannot complete a new lifecycle in subsequent hosts. Chapter 3 presents the Wolbachia levels in adult D. immitis with doxycycline treatment at different dosages. Chapter 4 explores gene expression alterations in D. immitis mf in the presence or absence of doxycycline treatment. This dissertation’s research investigated the impact of doxycycline on Wolbachia and D. immitis at different life cycle stages (mf, infective stage larvae, and adult), providing insights into the basic science of heartworm research and clinical practice in heartworm treatment.