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<i>Ascosphaera apis</i> infects exclusively bee larvae and causes chalkbrood, a lethal fungal disease that results in a sharp reduction in adult bees and colony productivity. However, little is known about the effect of <i>A. apis</i> infestation on the activities of antioxidant enzymes in bee larvae. Here, <i>A. apis</i> spores were purified and used to inoculate Asian honey bee (<i>Apis cerana</i>) larvae, followed by the detection of the host survival rate and an evaluation of the activities of four major antioxidant enzymes. At 6 days after inoculation (dpi) with <i>A. apis</i> spores, obvious symptoms of chalkbrood disease similar to what occurs in <i>Apis mellifera</i> larvae were observed. PCR identification verified the <i>A. apis</i> infection of <i>A. cerana</i> larvae. Additionally, the survival rate of larvae inoculated with <i>A. apis</i> was high at 1–2 dpi, which sharply decreased to 4.16% at 4 dpi and which reached 0% at 5 dpi, whereas that of uninoculated larvae was always high at 1~8 dpi, with an average survival rate of 95.37%, indicating the negative impact of <i>A. apis</i> infection on larval survival. As compared with those in the corresponding uninoculated groups, the superoxide dismutase (SOD) and catalase (CAT) activities in the 5- and 6-day-old larval guts in the <i>A. apis</i>–inoculated groups were significantly decreased (<i>p</i> < 0.05) and the glutathione S-transferase (GST) activity in the 4- and 5-day-old larval guts was significantly increased (<i>p</i> < 0.05), which suggests that the inhibition of SOD and CAT activities and the activation of GST activity in the larval guts was caused by <i>A. apis</i> infestation. In comparison with that in the corresponding uninoculated groups, the polyphenol oxidase (PPO) activity was significantly increased (<i>p</i> < 0.05) in the 5-day-old larval gut but significantly reduced (<i>p</i> < 0.01) in the 6-day-old larval gut, indicating that the PPO activity in the larval guts was first enhanced and then suppressed. Our findings not only unravel the response of <i>A. cerana</i> larvae to <i>A. apis</i> infestation from a biochemical perspective but also offer a valuable insight into the interaction between Asian honey bee larvae and <i>A. apis</i>.