Flavonoids are known to contribute to plant defense against herbivorous insects in a variety of systems, but their specific role in cassava (Manihot esculenta) resistance to the cassava mealybug (Phenacoccus manihoti) has remained unclear. Through expression profiling of nine flavonoid biosynthesis genes in high-resistance and high-susceptibility cassava varieties, chalcone synthase (CHS) was identified as the most differentially expressed candidate. Virus-induced gene silencing (VIGS) of CHS in the high-resistance variety C1115 led to a 67.5% reduction in CHS transcript levels, a 60.6% decrease in enzyme content, and a 29.7% decline in total flavonoid content. This was accompanied by the suppressed expression of downstream genes CHI and LAR. Silenced plants showed increased leaf damage and a higher pest index, effectively rendering the resistant phenotype susceptible. Furthermore, mealybugs feeding on these plants exhibited a 1.3-fold increase in their net reproductive rate. These results indicate that CHS-dependent flavonoid accumulation is one of the components required for resistance to P. manihoti in the high-resistance variety C1115, and identify CHS as a promising candidate target for marker-assisted breeding.