It is established that the immunogenicity of polysaccharides is enhanced by coupling them to carrier proteins. Cross reacting material (CRM₁₉₇), a nontoxic variant of diphtheria toxin (DT) is widely used carrier protein for polysaccharide conjugate vaccines. Conventionally, CRM₁₉₇ is isolated by fermentation of Corynebacterium diphtheriae C7 (β₁₉₇) cultures, which often suffers from low yield. Recently, several recombinant approaches have been reported with robust processes and higher yields, which will improve the affordability of CRM₁₉₇-based vaccines. Vaccine manufacturers require detailed analytical information to ensure that the CRM₁₉₇ meets quality standards and regulatory requirements. In the present manuscript we have described detailed structural characteristics of Escherichia coli based recombinant CRM₁₉₇ (rCRM₁₉₇) carrier protein. The crystal structure of the E. coli based rCRM₁₉₇ was found to be identical with the reported crystal structure of the C7 CRM₁₉₇ produced in C. diphtheriae C7 strain (Protein Data Bank (PDB) ID: 4EA0)_. The crystal structure of rCRM₁₉₇ was determined at 2.3 Å resolution and structure was submitted to the PDB with accession number ID 5I82. This is the first report of a crystal structure of E. coli derived recombinant CRM₁₉₇ carrier protein. Furthermore, the rCRM₁₉₇ was conjugated to Vi polysaccharide to generate Typhoid conjugate vaccine (Vi-rCRM₁₉₇) and its immunogenicity was evaluated in Balb/C Mice. The Vi-rCRM₁₉₇ conjugate vaccine was found to generate strong primary α-Vi antibody response and also showed a booster response after subsequent vaccination in mice. Overall data suggest that E. coli based recombinant CRM₁₉₇ exhibits structural and immunological similarity with the C7 CRM₁₉₇ and can be used as a carrier protein in conjugate vaccine development.