HIV-1 envelope (Env) glycoprotein is a trimer of heterodimer of gp120 and gp41, and derives from a trimeric glycoprotein precursor, gp160. Gp120 contains five conserved regions that are interspersed with 5 variable loop regions (V1-V5). Env variations in variable loop length and amino acid composition may associate with virus pathogenesis, virus sensitivity to neutralizing antibodies (nAbs) and disease progression. To investigate the role of each variable loop in Env function, we generated a panel of JRFL gp160 loop deletion mutants and examined the effects of each loop deletion on Env expression, Env cell surface display and Env-mediated virus entry into permissive cells. We found that deletion of V1 and V2 (ΔV1V2), or loop D (ΔlpD) abolished virus entry, the same effect as deletion of V3 (ΔV3), while deletion of V3 crown (ΔV3C) significantly enhanced virus assembly and entry. We further found that deletion of V4 (ΔV4) or V5 (ΔV5), or replacement of V4 or V5 with flexible linkers of the same lengths knocked out the receptor and coreceptor binding sites in gp120, but significantly enhanced the exposure of the N-trimer structure and the membrane proximal external region (MPER) in gp41. Although deletion of V4 or V5 did not affect Env expression, they negatively affected Env cell surface display, leading to the failure in virus assembly and subsequent entry. Taken together, we found that Env variable loops were indispensable for Env structural integrity and virus entry. Our findings may have implications for development of HIV-1 vaccine immunogens and therapeutics.