Background <p>Cassava (<i>Manihot esculenta</i> Crantz) is cultivated for its starchy root and mainly used as starch and biofuel feedstock in China. The red spider mite (<i>Tetranychus cinnabarinus</i> Boisduval) is one of the main insect pests reducing cassava yields and becoming more and more serious with regard to the increasing continuous cropping years in China.</p> Results <p>The results indicated that SC205 (4×) was more resistant to <i>T. cinnabarinus</i> infestation than SC205 (2×) according to the leaf damage ingestion, nutrient substance and secondary metabolite results. The <i>T. cinnabarinus</i> infestation triggered the expression of many genes and various metabolic processes reaction. Under the mite feeding stress, SC205 (2×) and SC205 (4×) shared 4494 and 5849 differentially expressed genes (DEGs) at 2 and 8 days, respectively. The DEGs were found enriched in the defense pathways flavonoid biosynthesis (map00941) and the flavone and flavonol biosynthesis (map00944), while,&#xa0;differentially accumulated metabolites (DAMs) were also found enhanced in flavonol biosynthesis (map00944) and phenylpropanoid biosynthesis pathway (map00940). Integrative analysis revealed that under 8-day pest hazards, both DEGs and DAMs in SC205 (2×) and SC205 (4×) were significantly co-enriched in several key pathways, including alpha-linolenic acid metabolism, ABC transporters, galactose metabolism, ascorbate and aldarate metabolism, alanine, aspartate and glutamate metabolism, and tyrosine metabolism. These findings suggest that carbohydrate metabolism and amino acid metabolism play crucial roles in cassava’s resistance to <i>T. cinnabarinus</i> infection.</p> Conclusions <p> Our study reveal the mechanisms of how cassava diploid and its autopolyploid in response to the feeding of <i>T. cinnabarinus</i> and provides data support for the precise analysis of cassava resistance and mite resistance breeding in further research.</p>