The carmine spider mite Tetranychus cinnabarinus is a major pest of crop and vegetable plants worldwide. Previous studies have shown that scopoletin is a promising acaricidal compound against Tetranychus cinnabarinus. However, the acaricidal mechanism of scopoletin remains unclear. In the present study, 12 full-length cDNAs of chitinase (CHIT) genes from Tetranychus cinnabarinus (designated TcCHITs) were cloned and characterized. Although TcCHITs were expressed throughout all life stages, their expression levels were significantly upregulated during the larval and nymphal stages. TcCHITs were downregulated 24 h after treatment with scopoletin and upregulated 24 h after treatment with diflubenzuron (DFB, a chitin synthesis inhibitor). Feeding double-stranded RNA effectively silenced TcCHIT transcription in Tetranychus cinnabarinus, thus increasing its susceptibility to scopoletin but reducing that to DFB. Meanwhile, TcCHIT silencing in larvae and adult resulted in an extremely low molting rate (7.3%) and high mortality rate (53.3%), respectively, compared with those in the control group. CHIT genes are closely related to arthropod survival, molting, and development in Tetranychus cinnabarinus, suggesting that acaricidal mechanisms of scopoletin and DFB may occur by inhibition and activation of CHIT gene expression, respectively. TcCHIT constitutes a possible target of scopoletin and DFB in Tetranychus cinnabarinus.