Tryptophan C-mannosylation is a co-translational modification unique to the metazoans and apicomplexan parasites. It is the only example of protein C-glycosylation reported to date and appears to play a role in protein folding, trafficking and/or stability. Our knowledge of the prevalence of this modification and its role in biology is limited, and hampered by a lack of tools for studying the modification. Here, we present a simple microbial system for the production of proteins with and without the modification, to facilitate characterisation of the effects of tryptophan mannosylation on protein stability and function. This system facilitates exploration of the C-mannosyltransferase substrate preference and catalytic machinery. The ability to readily produce proteins with this modification facilitated the generation and rigours characterisation of monoclonal antibodies capable of detecting C-mannosyl tryptophan. With these antibodies, and a robust expression system, we were able to identify the first small molecule capable of inhibiting C-mannosyltransferases: a susbtrate-mimicking peptide that is active in cells. Together, these tools provide a solid foundation for interrogating the role of C-mannosyl tryptophan in myriad biological systems.