Piwi-interacting RNAs (piRNAs) are a newly identified class of small noncoding RNAs. They are associated with chromatin organization, messenger RNA stability, and genome structure. Although the overexpression of piRNA-3312 in deltamethrin-susceptible (DS) strain of Culex pipiens pallens (L.) was observed in our previous large-scale transcriptome data, the roles of piRNA in insecticide resistance have not been clearly defined. The aim of the present study was to investigate how piRNA-3312 is involved in insecticide resistance. The lower expression level of piRNA-3312 in deltamethrin-resistant (DR) strain of Cx. pipiens pallens was confirmed by quantitative real time polymerase chain reaction (qRT-PCR). Overexpression of piRNA-3312 in the DR strain made the mosquitoes more sensitive to deltamethrin, whereas inhibiting the expression of piRNA-3312 in the DS strain made the mosquitoes more resistant to deltamethrin. Piwi-interacting RNA-3312 was also found to bind 3′ UTR (Untranslated Regions) of gut esterase 1 gene and could induce its degradation. In addition, knockdown of gut esterase 1 gene increased the sensitivity of DR strain to deltamethrin. In conclusion, we found that piRNA-3312 targeted the gut esterase 1 gene to negatively regulate the insecticide resistance. This finding facilitates the understanding of various functions of piRNAs and their association with insecticide resistance.