Microglia are key cells of the immune system in the central nervous system and are suggested to be deeply involved in the development of neurodegenerative diseases. It is well known that microglia have functional plasticity, with an inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Inhibition of choline transport in macrophages has been reported to suppress the secretion of inflammatory cytokines. However, the role of the choline transport system in regulating microglial M1/M2 polarization has not been fully elucidated to date. In this study, we investigated the mechanism of choline uptake in mouse microglial cell line SIM-A9, and its association with microglial M1/M2 polarization. Choline transporter-like protein 1 (CTL1) were highly expressed in SIM-A9 cells and were localized in the plasma membrane. Functional analysis of choline uptake demonstrated the existence of Na⁺-independent, pH-dependent, and intermediate-affinity transport systems. Choline uptake was concentration-dependently inhibited by hemicholinium-3 (HC-3). Expression of the mRNA of M1 microglia markers IL-1β and IL-6 was increased by LPS, and their effects were suppressed by choline deprivation and HC-3. In contrast, mRNA expression of the M2 microglial marker arginase-1 was increased by IL-4, and the effect was enhanced by choline deprivation and HC-3. Our results suggest that inhibition of CTL1-mediated choline uptake in microglia preferentially induces M2 microglia polarization, which is a potential therapeutic approach for inflammatory brain diseases.