Systemic inflammation can have devastating effects on the central nervous system via its resident immune cells, the microglia. One of the primary mediators of this inflammation is inflammasomes, multiprotein complexes that trigger a release of inflammatory proteins when activated. Melatonin, a hormone with anti-inflammatory effects, is an attractive candidate for suppressing such inflammation. In this study, we have investigated how melatonin alters the microRNA (miRNA) transcriptome of microglial cells. For that purpose, we have performed RNA sequencing on a lipopolysaccharide and adenosine triphosphate (LPS + ATP) induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation model in the N9 mouse microglial cell line, with and without melatonin pre-treatment. We have identified 136 differentially expressed miRNAs in cells exposed to LPS + ATP compared to controls and 10 differentially expressed miRNAs in melatonin pre-treated cells compared to the inflammasome group. We have identified miR-155-3p as a miRNA that is upregulated with inflammasome activation and downregulated with melatonin treatment. We further confirmed this pattern of miR-155-3p expression in the brains of mice injected intraperitoneally with LPS. Moreover, an overexpression study with miRNA-155-3p mimic supported the idea that the protective effects of melatonin in NLRP3 inflammasome activation are partly associated with miRNA-155-3p inhibition.