Microglial activation following peripheral nerve injury is crucial for neuropathic pain (NP) development; however, studies on time-specific and spatial characteristics of microglial transcriptome are scarce. Firstly, we comparatively analysed microglial transcriptome of different brain regions and multiple timepoints after nerve injury by analysing the gene expression profile of GSE180627 and GSE117320. Then, we performed a mechanical pain hypersensitivity test on 12 rat neuropathic pain models using von Frey fibres at various timepoints after nerve injury. To further explore the key gene clusters closely related to the neuropathic pain phenotype, we conducted a weighted gene co-expression network analysis (WGCNA) on the GSE60670 gene expression profile. Lastly, we performed a single-cell sequencing analysis on GSE162807 for identifying microglia subpopulations. We found that the trend of microglia’s transcriptome changes after nerve injury was that mRNA expression changes mainly occur early after injury, which is also consistent with phenotypic changes (NP progression). We also revealed that in addition to spatial specificity, microglia are also temporally specific in NP progression following nerve injury. The WGCNA findings revealed that the functional analysis of the key module genes emphasized the endoplasmic reticulum’s (ER’s) crucial role in NP. In our single-cell sequencing analysis, microglia were clustered into 18 cell subsets, of which we identified specific subsets of two timepoints (D3/D7) post-injury. Our study further revealed the temporal and spatial gene expression specificity of microglia in neuropathic pain. These results contribute to our comprehensive understanding of the pathogenic mechanism of microglia in neuropathic pain.