miRNAs play a key role in the autophagy process. In recent years, the emerging role of autophagy in regulating immune response has attracted increasing attention. Since then, specific miRNAs have also been found to play an immune function indirectly by modulating autophagy as well. This study proved that miR-23a could downregulate grass carp autophagy simultaneously by targeting ATG3 and ATG12. Besides, both ATG3 and ATG12 mRNA levels were increased in kidney and intestine after being infected by Aeromonas hydrophila; yet almost at the same time, miR-23a was decreased. Besides, we illustrated that grass carp miR-23a could affect antimicrobial capacity, proliferation, migration, and antiapoptotic abilities of CIK cells. These results indicate that miR-23a was related to grass carp autophagy and plays an important role in antimicrobial immunity through targeting ATG3 and ATG12, which provides important information on autophagy-related miRNAs about the defense and immune mechanisms against pathogens in teleost.