Shiga toxin-producing Escherichia coli (STEC) are widely detected in raw milk products intended for hu-man consumption. Although STEC are a worldwide public health problem, the pathogenicity of STEC in cheese remains unclear. In fact, bacterial association with compounds in raw milk cheeses could reduce their pathogenicity. A previous study showed the association of 2 STEC strains with raw milk cream in a natural creaming assay. Different concentrations of each strain were required to saturate the cream. In this study, we hypothesized that all STEC strains could be associated with milk fat globules (MFG) in raw milk and that the bacterial load required for saturation of the cream is serotype dependent. We evaluated the affinity of STEC strains belonging to the O157:H7, O26: H11, and O103:H2 serotypes for bovine raw milk cream and ana-lyzed saturation of the cream layer by natural creaming assay. We used 12 STEC strains and 3 strains belonging to another pathotype to assess the effects of serotypes on this phenomenon. We performed sucrose density gra-dient centrifugation assays with 2 STEC model strains to confirm the results obtained by natural creaming. The localization of STEC within MFG-enriched creams was observed by confocal and electron microscopy. We recovered approximately 10 times more STEC from the cream layer after natural creaming than from raw bovine milk. The concentration of STEC required to saturate the cream layer (the saturation concentration) was estimated for each strain by nonlinear regression, highlighting a strain and serotype effect. Moreover, the concentration of STEC in the cream was milk fat level dependent. However, even in nonsaturating conditions, a high level of STEC was still present in the aque-ous phase, after fat separation. Thus, natural creaming should not be used as the sole preventive measure to remove STEC from naturally contaminated raw milk. The results of our study suggest that cream saturation is a complex mechanism, most likely involving specific interactions between STEC and raw MFG.