ABSTRACT When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus , c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N -acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression. IMPORTANCE Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus . We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems.