A single mutation in the envelope protein (E1-A226V) of Chikungunya virus (CHIKV) significantly affects its vector specificity and epidemic potential. This mutation, found in the 2005–2006 epidemic on Reunion Island, increased CHIKV infectivity in Aedes albopictus mosquitoes, leading to more efficient viral dissemination and transmission to suckling mice. While the mutation slightly reduced CHIKV infectivity in Aedes aegypti mosquitoes, it slightly increased transmission to suckling mice. The mutation also increased CHIKV's dependence on cholesterol in the mosquito cell membrane, which is associated with increased fitness in Aedes albopictus. These findings suggest that the E1-A226V mutation allows CHIKV to adapt to Aedes albopictus, a vector not typically associated with CHIKV transmission, thereby contributing to the epidemic. The mutation's effect on vector specificity and transmission has important implications for the geographic spread of CHIKV, as Aedes albopictus is widely distributed in Europe and the Americas. The study highlights how a single amino acid substitution can significantly influence viral fitness and transmission, providing insights into how viruses may establish transmission cycles in new areas. The results also suggest that the E1-A226V mutation may have contributed to the maintenance and scale of the epidemic on Reunion Island.A single mutation in the envelope protein (E1-A226V) of Chikungunya virus (CHIKV) significantly affects its vector specificity and epidemic potential. This mutation, found in the 2005–2006 epidemic on Reunion Island, increased CHIKV infectivity in Aedes albopictus mosquitoes, leading to more efficient viral dissemination and transmission to suckling mice. While the mutation slightly reduced CHIKV infectivity in Aedes aegypti mosquitoes, it slightly increased transmission to suckling mice. The mutation also increased CHIKV's dependence on cholesterol in the mosquito cell membrane, which is associated with increased fitness in Aedes albopictus. These findings suggest that the E1-A226V mutation allows CHIKV to adapt to Aedes albopictus, a vector not typically associated with CHIKV transmission, thereby contributing to the epidemic. The mutation's effect on vector specificity and transmission has important implications for the geographic spread of CHIKV, as Aedes albopictus is widely distributed in Europe and the Americas. The study highlights how a single amino acid substitution can significantly influence viral fitness and transmission, providing insights into how viruses may establish transmission cycles in new areas. The results also suggest that the E1-A226V mutation may have contributed to the maintenance and scale of the epidemic on Reunion Island.