Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria. While the benefits of LGG treatment in gastrointestinal disorders and immunomodulation are well-documented, functional genomics research of this bacterium has only recently been initiated. In the present study, a 2-D DIGE approach was used for the quantitative analysis of growth media-dependent changes in LGG protein abundance. Proteins were isolated from cells grown in industrial-type whey-based medium or in rich laboratory medium for subsequent 2-D DIGE. The analysis revealed patterns of protein abundance unique to each growth condition. In total, 196 quantitatively altered protein spots (at least 1.5-fold change in relative abundance, p < 0.05) representing approximately 13% of all protein spots in the gel were detected. From these protein spots, 157 were identified by mass spectrometry and were found to represent 100 distinct gene products. Collectively, these data show that growth of LGG in whey medium increased the relative abundance of proteins involved in purine biosynthesis, galactose metabolism, and fatty acid biosynthesis. In comparison, growth of LGG in laboratory medium resulted in an increase in the amount of proteins involved in translation and the general stress response, as well as pyrimidine and exopolysaccharide biosynthesis. Moreover, several enzymes of the proteolytic system of LGG demonstrated growth medium-dependent production. The present study demonstrates the fundamental effects of culture conditions on the proteome of LGG, which are likely to affect the functionality and characteristics of its use as a probiotic.