The lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a monophagous and dangerous pest of mulberry that has been recently observed in Guilan province, northern Iran. In this study, the crude methanol extract of sweet wormwood Artemisia annua L. (Asterales: Asteracaea) was investigated on toxicity, biological and physiological characteristics of this pest under controlled conditions (24 ± 1 °C, 75 ± 5% RH, and 16:8 L:D photoperiod). The effect of acute toxicity and sublethal doses on physiological characteristics was performed by topical application. The LC₅₀ and LC₂₀ values on fourth instar larvae were calculated as 0.33 and 0.22 gram leaf equivalent/ mL, respectively. The larval duration of fifth instar larvae in LC₅₀ treatment was prolonged (5.8 ± 0.52 days) compared with the control group (4.26 ± 0.29 days). However larval duration was reduced in the LC₂₀ treatment. The female adult longevity in the LC₅₀ dose was the least (4.53 ± 0.3 days), while longevity among controls was the highest (9.2 ± 0.29 days). The mean fecundity of adults after larval treatment with LC₅₀ was recorded as 105.6 ± 16.84 eggs/female, while the control was 392.74 ± 22.52 eggs/female. The percent hatchability was reduced in all treatments compared with the control. The effect of extract in 0.107, 0.053, 0.026 and 0.013 gle/mL on biochemical characteristics of this pest was also studied. The activity of α-amylase and protease 48 hours post—treatment was significantly reduced compared with the control. Similarly lipase, esterase, and glutathione S-transferase activity were significantly affected by A. annua extract.