Heavily glycosylated secreted mucin MUC5AC, by the virtue of its cysteine-rich repeats, can form inter- and intramolecular disulfide linkages resulting in complex polymers, which in turn craft the framework of the polymeric mucus gel on epithelial cell surfaces. MUC5AC is a molecule with versatile functional implications including barrier functions to epithelial cells, host-pathogen interaction, immune cell attraction to sites of premalignant or malignant lesions and tumor progression in a context-dependent manner. Differential expression, glycosylation and localization of MUC5AC have been associated with a plethora of benign and malignant pathologies. In this era of robust technologies, overexpression strategies and genetically engineered mouse models, MUC5AC is emerging as a potential diagnostic, prognostic and therapeutic target for various malignancies. Considering the clinical relevance of MUC5AC, this review holistically encompasses its genomic organization, domain structure, glycosylation patterns, regulation, functional and molecular connotation from benign to malignant pathologies. Furthermore, we have here explored the incipient and significant experimental tools that are being developed to study this structurally complex and evolutionary conserved gel-forming mucin.