This meta-analysis aimed to investigate the effect of dietary fatty acid (FA) profile on milk fat production and FA profile in dairy cows. The study also aimed to develop prediction models using a meta-regression approach. The database included 217 peer-reviewed articles on lactating dairy cows (n = 12,892), consisting of 515 treatment means. Effect size was assessed using the raw mean differences between diets with supplementary lipid sources and those without. Subgroup analyses were employed to assess heterogeneity. Diets rich in saturated FA (SFA) increased milk fat production and proportion, while reducing de novo FA in milk. Diets high in monounsaturated FA and polyunsaturated FA decreased mixed FA in milk. Most lipid-supplemented diets increase preformed FA in milk, except those rich in SFA. Prediction models were developed using meta-regression. Key predictors of milk fat production included neutral detergent fiber (NDF), dietary myristic acid, and milk production. Milk fat proportion was best predicted by dietary unsaturated FA, NDF, and forage. De novo FA in milk was predicted by dry matter intake (DMI) and dietary FA, while preformed FA was predicted by DMI, dietary oleic and linoleic acids. In conclusion, this study emphasizes the importance of the dietary FA profile in evaluating the effects of lipids on milk fat production and FA profile. Accurate and precise predictions of milk fat production, proportion, and FA profile can be achieved by considering cow production and dietary characteristics.