Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry, and the S protein glycosylation plays key roles in altering the viral binding/function and infectivity. However, the molecular structures and glycan heterogeneity of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis by conventional bottom-up glycoproteomic approaches. Here, we report the complete structural elucidation of intact O-glycan proteoforms through a hybrid native and denaturing top-down mass spectrometry (MS) approach employing both trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR)-MS. Native top-down TIMS-MS/MS separates the protein conformers of the S-RBD to reveal their gas-phase structural heterogeneity, and top-down FTICR-MS/MS provides in-depth glycoform analysis for unambiguous identification of the glycan structures and their glycosites. A total of eight O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that this hybrid top-down MS approach can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants as well as other O-glycoproteins in general.