The requirement of the exopolysaccharide amylovoran for Erwinia amylovora pathogenesis is well documented. However, regulation of amylovoran biosynthesis has not been comprehensively studied. We have previously reported that amylovoran production is strain-dependent in E. amylovora isolates. We have also demonstrated that amylovoran biosynthesis is mainly controlled by the RcsBCD phosphorelay two-component system, which is essential for virulence. In this study, we have systematically characterized a network of regulatory genes as well as various environmental conditions such as pH, temperature, and carbon sources in regulating amylovoran production in vitro. We found that global regulators such as H-NS and accessory proteins of the Rcs system such as RcsF and DjlA negatively regulated amylorovan production in E. amylovora strain Ea1189. In addition, a hypothetical gene (herein named amyR) was also identified that negatively regulated amylovoran production. Our results also indicated that E. amylovora strains produced more amylovoran at high temperature (32°C) or neutral pH (7.0) than at low temperature (22 or °) or acidic pH (5.5). Furthermore, an rcsC mutant strain did not produce higher levels of amylovoran at high temperature than at low temperature, indicating that RcsC may be involved in thermo-regulation of amylovoran production. These results indicate that regulation of amylovoran production is very complicated in E. amylovora and suggest the presence of regulatory networks governing the expression of amylovoran biosynthesis genes.