Abstract Background Microglial cultures comprise a critically important model system for investigating inflammatory mechanisms in almost all CNS disorders. Mild trypsinization and shaking are the two most commonly used methods to isolate primary microglia from mixed glial cultures. In this study, we characterized and compared microglia obtained using these two methods. Methods Primary rat microglia cultures were prepared from cerebral cortices of 1–2-day-old neonatal Sprague-Dawley rats. After achieving confluency at about 14 days in vitro, microglia were isolated from mixed glial cultures via either mild trypsinization or shaking. The purity of microglia was estimated by flow cytometry. Quantitative real-time PCR was used to measure mRNA expression. TNFα, IL-1β, IL-10, and IGF-1 in cell culture supernatant were measured using ELISA kits. Phagocytic function was assessed using fluorescein-labeled Escherichia coli K-12 BioParticles. Results Mild trypsinization generated a higher yield and purity than shaking. Microglia isolated by mild trypsinization appeared to be in a quiescent state with ramified morphology. Microglia isolated by shaking showed a more heterogenous morphology, including cells with rounded shapes suggestive of activation. Compared with shaking, microglia isolated by trypsinization also had lower baseline phenotype markers (iNOS, CD86, CD206, and arginase 1) and lower levels of cytokines (TNFα, IL-1β, IL-10, and IGF-1) as well as reduced phagocytic capability. Both methods yielded microglia that were responsive to various stimuli such as IL-4, lipopolysaccharide (LPS), or interferon-γ (IFNγ). Although stimulated patterns of gene expression and cytokine release were generally similar, there were also significant differences in terms of absolute response. LPS treatment induced significantly higher levels of TNFα and IL-10 in microglia isolated by mild trypsinization versus shaking. IFNγ induced a lower response in TNFα in microglia obtained by mild trypsinization versus shaking. Conclusions Our results suggest that isolating microglia with the shaking method may induce slight activation even at baseline, and this may affect stimulus responses in subsequent experiments. Caution and attention should be warranted when choosing isolation protocols for primary microglia cultures.