Due to a great interest in application of bovine MFGM as a nutraceutical (J. Dairy Sci. 88:2289–2294, 2005), we developed a new protocol for isolation of large amounts of MFGM from buttermilk. The protocol includes the following steps: precipitation of casein and MFGM at pH 4.6, treatment of the precipitated complex casein-MFGM with sodium phosphate at pH 4.6–8.0 or with polyphosphate (Calgon) at pH 7.1 and differential centrifugation (1000–2000 × g to get a pellet of casein and 32,000 × g (Strator centrifuge) to collect a pellet of MFGM. Maximum dissociation of MFGM from casein was achieved at pH 7.1–7.4. Yield of MFGM from 1 L of buttermilk treated with sodium phosphate at pH 7.2 was 789 mg of protein and treatment with Calgon at pH 7.1 also provided a high yield of MFGM, 725 mg/L. Treatment of precipitated casein with 0.1 M Na-citrate or Na-oxalate at pH 7.1 led to the isolation of MFGM in amount of 234 mg/L and 106 mg/L, correspondingly. Treatment of precipitated casein with 0.1 M NaCl or 0.1 M Na₂SO₄ did not produce a noticeable amount of MFGM. Recovery of MFGM from buttermilk allowed us to suggest that MFGM in buttermilk is present in association with casein through Ca- bridges between phospholipids of MFGM and phosphate groups of casein, primarily with k-casein as a peripheral protein of casein micelles. For production of large amount of MFGM on industrial scale the use of continuous-flow centrifuge and microfiltration were suggested. Baemek Advanced Technology supported this work.