Microcystins (MCs) are the most widely distributed cyanobacterial toxins that can exert adverse effects on aquatic organisms, but aside from the study of the harmful effect of cyanobacterial blooms, little is known about the effect of released MCs on the growth and development of chironomid larvae. To assess the harmful effect and the toxic mechanism of MCs on midges, the life-history traits, intestinal microbiota, and transcriptome of Chironomus pallidivittatus were analyzed after chronic exposure to 30 mu g/L of MC-LR. Exposure inhibited larvae body length by 35.61% and wet weight by 21.92%, increased emergence time of midges, damaged mitochondria in the intestine, promoted oxidative stress, dysregulated lipid metabolism of chironomid larvae, and increased detoxification enzymes glutathione S-transferase (GST) and superoxide dismutase (SOD) by 32.44% and 17.41%, respectively. Exposure also altered the diversity and abundance of the intestinal microbiota, favoring pathogenic and MC degradation bacteria. RNA sequencing identified 261 differentially expressed genes under MC-LR stress, suggesting that impairment of the peroxisome proliferator-activated receptor signaling pathway upregulated fatty acid biosynthesis and elongation to promote lipid accumulation. In addition, exposure-induced detoxification and antioxidant responses, indicating that the chironomid larvae had the potential ability to resist MC-LR. To our knowledge, this is the first time that lipid accumulation, oxidative stress, and detoxification have been studied in this organism at the environmentally relevant concentration of MC-LR; the information may assist in ecological risk assessment of cyanobacterial toxins and their effects on benthic organisms.