A novel method to improve the proliferation activity of C₂C₁₂ cells by the bovine milk fat globule membrane (MFGM) protein was established in this study. The MFGM protein was extracted and isolated into 4 fractions using an electric cream separator, and purified by a cellulose DEAE-52 column. Fraction 2 accounted for 57.8% of the total MFGM protein, and was used in the following study. The MTT assay showed that it induced cell proliferation activity, increased the cell survival rate and the cell number using flow cytometry and fluorescence microscopy analysis. There were only subtle changes in the morphology as observed using confocal scanning laser microscopy, but the number of mitochondria was significantly increased as observed using transmission electron microscopy analysis. Furthermore, the mRNA expression of MyoD, cyclin D1, p70S6K and mTOR was up-regulated as determined utilizing the quantitative real-time PCR assay, and the activation of Akt and mTOR phosphorylation was up regulated as determined using the Western blot assay. The main protein in fraction 2, assayed by 1-D gel electrophoresis and MALDI TOF-TOF, was identified as milk fat globule-EGF factor 8, the content was 65.6% of the total protein in fraction 2. The results elucidate a new molecular mechanism of the MFGM protein fraction 2: the activation of the Akt signal pathway in promoting cell proliferation.