Both 20-hydroxyecdysone (20E) and miRNAs have multiple functions in the regulation of various physiological processes in insects. However, little is known about the interaction between 20E and miRNAs. In this study, six small RNA libraries were constructed from the adult Galeruca daurica treated with 20E and dimethyl sulfoxide (DMSO), respectively. Using small RNA sequencing, a total of 183 miRNAs, including 140 known and 43 novel miRNAs, were identified. Compared with the control (DMSO), 52 miRNAs (21 up-regulated and 31 down-regulated) were significantly differentially expressed after 20E treatment. The KEGG and GO analysis of the predicted genes targeted by 20E-responsive miRNAs indicate that 20E may influence the metabolic change during reproductive diapause in G. daurica via regulating miRNAs.