ABSTRACT: Milk fat content is a polygenic commercially important quantitative trait in ruminants. In recent decades, an increasing number of genes involved in milk fat synthesis have been identified through GWAS and validated using functional assays, such as gene knockdown or overexpression. However, functional validation assays have been limited to a small number of related genes, yielding insufficient data to fully understand the biological processes in milk fat synthesis. To provide a comprehensive evaluation of the genes involved in milk fat synthesis, we performed a meta-analysis of 1,395 effect sizes from 81 publications, which included 137 genes, spanning 4 ruminant species. In the studies, analyzed knockdown/overexpression of candidate genes significantly reduced/increased target gene (protein) and related gene (protein) expression, and these effects were consistent across species. However, the effects of knockdown/overexpression of candidate genes across the different pathways of milk fat synthesis varied between species. Pathways related to milk fat synthesis, SREBPp, PPAR, JAK-AKT, and the Insulin pathway, exhibited the largest effects on the synthesis of triglyceride, lipid droplet, cholesterol, and UFA, respectively. Key genes in these pathways, SREBP (SREBP1, SREBF1), PPAR (PPARA, PPARD, PPARG), JAK2, STAT5α, and INSIG (INSIG1 and INSIG2), also have a significant effect on regulating the formation of triglyceride, cholesterol, lipid droplets, and UFA, respectively. Overall, our findings indicated that milk fat synthesis is regulated by multiple pathways and many different genes. Further studies are needed to confirm these findings and to understand the mechanisms underlying species- and pathway-specific responses during milk fat synthesis.