Advances in HIV-1 envelope glycoprotein (Env) design generate native-like trimers and high-resolution clade A, B, and G structures and elicit neutralizing antibodies. However, a high-resolution clade C structure is critical, as this subtype accounts for the majority of HIV infections worldwide, but well-ordered clade C Env trimers are more challenging to produce due to their instability. Based on targeted glycine substitutions in the Env fusion machinery, we defined a general approach that disfavors helical transitions leading to post-fusion conformations, thereby favoring the pre-fusion state. We generated a stabilized, soluble clade C Env (16055 NFL) and determined its crystal structure at 3.9 Å. Its overall conformation is similar to SOSIP.664 and native Env trimers but includes a covalent linker between gp120 and gp41, an engineered 201-433 disulfide bond, and density corresponding to 22 N-glycans. Env-structure-guided design strategies resulted in multiple homogeneous cross-clade immunogens with the potential to advance HIV vaccine development. Display omitted •Structure-guided design generates an HIV clade C Env crystal structure at 3.9 Å•Cross-clade Env comparison discloses overall structural and N-glycan conservation•The NFL structure reveals stabilizing TD contacts and the 201C-A433C disulfide CC•NFL Env redesign permits the generation of immunogens derived from clades A, B, and C The majority of HIV-1 infections worldwide emanate from subtype C strains. Guenaga et al. describe the 3.9 Å crystal structure of a stabilized subtype C native, flexibly linked (NFL) Env and multiple structure-guided design strategies that permit the generation of Env immunogens from diverse HIV strains.