Background: Cladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood-brain barrier suggesting a potential effect on CNS resident cells. Objective: We examined, if CdA modifies the phenotype and function of naïve and activated primary mouse microglia, when applied in different concentrations including 0.1-1 µM that putatively overlaps human CSF concentrations. Methods: Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti-inflammatory IL-4 were co-treated with different concentrations of CdA for 24 hours. Viability was assessed by MTT assay. Phagocytotic ability and morphology were examined by flow cytometry, and random migration by IncuCyte Zoom and TrackMate. Change in gene expression was examined by qPCR, and protein secretion by Meso Scale Discovery. Results: LPS and IL-4 upregulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0.1-1 µM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 µM increased the IL-4-induced gene expression of Arg1 and LPS-induced expression of IL-1beta, TNF, iNOS, and Arg1, but protein secretion remained unaffected. CdA 10 µM potentiated the increased expression of anti-inflammatory TNFR2 but not TNFR1 induced by LPS. Conclusion: Microglia acquire a less activated phenotype when treated with 0.1–1 µM CdA that putatively overlaps human CSF concentrations. This may be related to the upregulated gene expression of DCK upon activation and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.