Buttermilk is the aqueous phase obtained after churning of cream into butter. Its composition is similar to that of skim milk except for higher proportion of milk fat globule membrane (MFGM) components. These components seem to interfere with rennet coagulation of milk. The purpose of the present study was to separate buttermilk serum into 3 fractions using centrifugation and determine the effect of each fraction on milk rennet-induced coagulation. Buttermilk and pasteurized skim milk (control) were centrifuged (31,000 g, 34°C, 1 h). Three distinct layers of the supernatants were collected: low-density opalescent (LDO), clear (CL) and high-density opalescent (HDO) layers. Protein and fat in these fractions were characterized by gel electrophoresis (SDS-PAGE) and estimation of phospholipid content by colorimetric phosphorus assay after fat extraction by the Mojonnier procedure. Casein micelles from skim milk were collected by centrifugation (31,000 g, 20°C, 1 h), redispersed in the different fractions and used to monitor rennet aggregation and coagulation kinetics using dynamic light scattering and rheology. MFGM protein and residual casein micelles were mainly concentrated in the HDO fraction. This fraction from buttermilk contained 3 times more MFGM protein than the same fraction from skim milk, but 35% less casein. Phospholipids were also concentrated in this fraction, 200 mg/100 g and 17 mg/100 g, representing 43 and 39% total fat of this fraction in buttermilk and skim milk, respectively. All buttermilk serum fractions, especially HDO, showed negative effect on rennet coagulation kinetics. Compared with the results obtained with their respective CL fraction (which is the LDO and HDO-depleted fraction), buttermilk HDO slowed down the aggregation rate (−62%) while skim milk HDOL increased it (+42%). In addition, the growth of casein aggregates was rapidly stopped in presence of buttermilk HDO fraction. This suggests that components in this fraction from buttermilk interfere with the aggregation of renneted casein micelles and more investigation is underway to determine the role of MFGM components.