29 years have passed since the discovery of bis-(3′,5′) cyclic di-guanylate (c-di-GMP) which is a key bacterial second messenger involved in the regulation of many crucial bacterial processes including biofilm formation and disappearing, cell motility, virulence, cell cycle controlling, cell differentiation and aggregation as well. c-di-GMP plays an irreplaceable role in biosynthesis and transportation of bacterial extracellular polysaccharides (EPS), which are major component of bacterial biofilm. Currently, due to its wide range of application in medicine, food processing, agriculture industries and environmental protection, it has gained more attention from medical and nutritional biochemistry researchers. In this paper we first reviewed recent achievements on crystal structure of c-di-GMP metabolic enzymes and its multiple receptor protein of c-di-GMP with emphasis on its in vivo concentration regulation; then combined with our researches on the regulation of curdlan, biosynthesis by c-di-GMP in Agrobacterium sp. ATCC31749, we further focus on the advancement of c-di-GMP regulation on the biosynthesis and transportation of bacterial extracellular polysaccharides (EPS), such as cellulose, alginate, poly-N-acetylglucosamine (PNAG) and curdlan as well, which were widely used in industrial, pharmaceutical and food applications.