The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.