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In the present study, the toxicity and physiological disorders of the essential oil isolated from <i>Artemisia annua</i> flowers were assessed against one of the main insect pests of mulberry, <i>Glyphodes pyloalis</i> Walker, announcing one of the safe and effective alternatives to synthetic pesticides. The LC₅₀ (lethal concentration to kill 50% of tested insects) values of the oral and fumigant bioassays of <i>A. annua</i> essential oil were 1.204 % W/V and 3.343 μL/L air, respectively. The <i>A. annua</i> essential oil<i>,</i> rich in camphor, artemisia ketone, β-selinene, pinocarvone, 1,8-cineole, and α-pinene, caused a significant reduction in digestive and detoxifying enzyme activity of <i>G. pyloalis</i> larvae. The contents of protein, glucose, and triglyceride were also reduced in the treated larvae by oral and fumigant treatments. The immune system in treated larvae was weakened after both oral and fumigation applications compared to the control groups. Histological studies on the midgut and ovaries showed that <i>A. annua</i> essential oil caused an obvious change in the distribution of the principal cells of tissues and reduction in yolk spheres in oocytes. Therefore, it is suggested that the essential oil from <i>A. annua</i> flowers, with wide-range bio-effects on <i>G. pyloalis</i>, be used as an available, safe, effective insecticide in the protection of mulberry.