We have identified a mutant human immunodeficiency virus type 1 (HIV-1) CRF01_AE that contains a single nucleotide mutation in gag gene from 4 HIV-1 seronegative drug users in Thailand. We found A to G mutation at the nucleotide position 75 of gag p17 gene (A75G) not changing the amino acid sequence. The mutant HIV-1 molecular clones were examined for their replication capability. Although the mutation dramatically reduced the level of virion production, it did not affect the amounts of viral protein synthesis within the transfected cells. In addition, this mutation did not affect the levels of Gag polyproteins. Furthermore, electron microscopic examinations have revealed a dramatic reduction of the virion production and perturbation of viral morphogenesis at the cytoplasmic membrane. These results indicate that the A75G mutation is attributable to the long-term sero-negativity of individuals at high risk of HIV-1 infection and suggest a novel mechanism that regulates HIV production.