The aim of this study was to evaluate the effect of conjugated linoleic acid (CLA) on milk fat globule (MFG) size and the ruminal microbiome of goats. Twenty-four mid-lactation Saanen dairy goats weighing 49 ± 4.5 kg (168 ± 27 d in milk, 1.2 ± 0.1 kg milk/d, 2-3 years old) were randomly divided into four groups-a control (CON) group, which was fed a basal diet, and three CLA supplementation groups, in which 30 g CLA (low-dose group, L-CLA), 60 g CLA (medium-dose group, M-CLA), or 90 g CLA (high-dose group, H-CLA) was added to the basal diet daily. The experiment lasted for 21 days, during which time goat milk was collected for composition and MFG size analysis. On day 21 of feeding, ruminal fluid was collected from the CON and H-CLA groups for analysis of the changes in microorganismal abundance. The results showed that CLA supplementation did not affect milk production, milk protein, or lactose content in the dairy goats (p > 0.05), but significantly reduced the milk fat content (p < 0.01) compared with the CON group. The CLA supplementation significantly decreased the D[3,2] and D[4,3] of the MFGs in a dose-dependent manner (p < 0.01). Moreover, dietary CLA inclusion increased the proportion of small-sized MFGs and decreased that of large-sized ones. The results of 16S rRNA gene sequencing showed that CLA-induced milk fat depression in dairy goats was accompanied by significant changes in the relative abundance of ruminal bacterial populations, most of which belonged to the Firmicutes and Bacteroidetes phyla. The relative abundance of Rikenellaceae_RC9_gut_group and Prevolellaceae_UCG-003 in Bacteroidetes and UCG-002, Succiniclasticum, and norank_f__norank_o__Clostridia_vadinBB60_group in Firmicutes was significantly higher in the CON group than in the H-CLA group. In contrast, the relative abundance of norank_f__UCG-011, norank_f_Eubacterium_coprostanoligenes_group, unclassified_f__Lachnospiraceae, and UCG-001 in Firmicutes and norank_f__Muribaculaceae in Bacteroidetes was significantly higher in the H-CLA group than in the CON group. Correlation analysis showed that the milk fat content was negatively correlated with the relative abundance of some bacteria, including members of Firmicutes and Bacteroidetes. Similarly, MFG size (D[3,2] and D[4,3]) was negatively correlated with several members of Firmicutes and Bacteroidetes, including Lachnospiraceae, norank_f__UCG-011, UCG-001, norank_f__Eubacterium_coprostanoligenes_group (Firmicutes), and norank_f__Muribaculaceae (Bacteroidetes), while positively correlated with the relative abundance of some members of Firmicutes and Bacteroidetes, including Mycoplasma, Succiniclasticum, norank_f__norank_o__Clostridia_vadinBB60_group, UCG-002 (Firmicutes), and Rikenellaceae_RC9_gut_group (Bacteroidetes). Overall, our data indicated that CLA treatment affected milk fat content and MFG size in dairy goats, and these effects were correlated with the relative abundance of ruminal bacterial populations. These results provide the first evidence to explain the mechanism underlying diet-induced MFG from the perspective of the ruminal microbiome in dairy goats.