The milk fat globule membrane (MFGM) that surrounds fat globules in milk is a natural source of sphingolipids, phoshpholipids and proteins with defined anticancer properties. Dairy processing operations can affect the structure and composition of MFGM, potentially influencing its anticancer activity. The aim of this project was to determine if anticancer activity can be attributed to sweet and fermented buttermilks which contain fragments of MFGM and examine if different milk processing operations (separation, washing, heating and drying) may influence the antiproliferative activity of resultant buttermilks. After 3 days of incubation sweet pasteurized buttermilk at the concentration of 0.38 mg total solids/ml significantly (P ≤ 0.001) inhibited growth of SW480 human colon cancer cells by 97.5% but had no toxic effect on FHC human normal colon cells as determined by the acid phosphatase cytotoxicity assay. Antiproliferative activity was lost after spray drying of buttermilk but was retained after heat treatment and freeze and spin drying. In contrast to natural buttermilks, fermented buttermilks inhibited growth to a lesser extent. Analysis of mitochondrial permeability and phoshatidylserine exposure in cells using flow cytometry suggest induction of apoptosis as a biological mechanism involved in the inhibitory effect exerted by buttermilk fractions on cancer cell growth.