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【Objective】The key target genes related to late pregnancy, lactation and fecundity in muscle tissue of Holstein cattle were screened, with a view to providing a theoretical basis for further study of gene function.【Method】The dataset GSE62159 from the GEO database on gene expression and fecundity in the muscle of Holstein cattles during late pregnancy, early lactation, and mid lactation were selected. The weighted gene co-expression network analysis (WGCNA) method was used for gene co- expression analysis, and the obtained modules were associated with late pregnancy, mid lactation and fecundity traits. The genes with connectivity in the top 30 of each module were selected as the Hub genes. The module was analyzed by protein-protein interaction (PPI) network with String website. The gene with the top 30 node connection degree value in PPI network was selected as the core gene, and intersected with Hub gene to obtain the key target genes related to different physiological periods and fecundity in the muscle.【Result】A total of 13 modules were obtained, of which the Green and Blue modules were related to late pregnancy and mid-lactation periods respectively, and the Magenta module was related to fecundity. The functional enrichment analysis of genes in each target module showed that the main functions of genes related to the late pregnancy were substance metabolism, energy metabolism, protein synthesis and secretion, body response to oxidative stress and neurodegeneration; the functions of genes related to mid-lactation included maintenance of stem cell population, protein synthesis and secretion, antigen processing and presentation and occurrence of many diseases; while those related to fecundity included angiogenesis, intrauterine embryonic development and actin filament binding, etc. Three key genes related to late pregnancy, one key gene related to mid-lactation and eight key genes related to fecundity were screened in the muscle of Holstein cattle.【Conclusion】The genes related to and late pregnancy in the muscle of Holstein cattle were identified as EIF5A, ACO2 and EEF1G, and the gene related to mid-lactation as EIF4A2, and the genes related to fecundity as ITGB1, MYH9, TLN1, CAV1, COL4A1, COL4A2, FLNA and HSPG2.