Cytidine 5'-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.