Sitagliptin, used for type-2 diabetes, inhibits dipeptidyl peptidase 4 (DPP-4), thus increasing insulin secretion. We found that sitagliptin protects the retina of diabetic animals, including type-1 diabetic animals, inhibiting nitrosative stress and neuroinflammation. We also showed that microglia-mediated neuroinflammation triggers retinal degeneration. Here, we investigated whether sitagliptin effects can be due to a direct inhibition of microglia reactivity, mediated by DPP-4 blockade, or if pleiotropic mechanisms can be involved. Primary mixed and organotypic rat retinal cell cultures and BV-2 microglia were exposed to lipopolysaccharide (LPS) for 24h, in the absence/presence of sitagliptin. The immunoreactivity (IR) of iNOS in CD11b+ cells and nitrites levels were quantified. Microglia morphology was assessed in organotypic cultures. In BV-2 cells, DPP-4 expression, and iNOS, IL-1-beta and TNF-alpha levels were evaluated by RT-PCR or Western Blot, and the phagocytic efficiency using fluorescent microbeads. Dpp4 deletion was generated using CRISPR/Cas9. LPS increased iNOS IR in microglia in mixed and organotypic cultures. Sitagliptin significantly inhibited these effects and the nitrites levels. LPS induced microglia morphology changes, which were prevented by sitagliptin. BV-2 cells express DPP-4. LPS increased iNOS, IL-1 beta and TNF levels, and phagocytic efficiency of BV-2 cells, all inhibited by sitagliptin. Importantly, Dpp4 deletion abrogated the inhibitory effect of sitagliptin on phagocytic efficiency and IL-1 beta levels. In conclusion, sitagliptin exerts direct anti-inflammatory effects in microglia by inhibiting DPP-IV expressed in these cells. Additional experiments are needed to clarify if pleiotropic mechanisms can be also involved. Support: FCT-Portugal: UIDB/04539/2020, UIDP/04539/2020 and PTDC/NEU-OSD/1113/2012. COMPETE-FEDER (POCI-01-0145-FEDER-007440). FMUC:FA0911.