In Mali, malaria vector control relies mostly on long-lasting insecticidal nets and indoor residual spraying (IRS). From 2008 to 2016, an IRS program was implemented in the district of Koulikoro. After a significant reduction in malaria indicators, IRS was stopped in 2016. This study evaluated the effect of IRS withdrawal on entomological parameters of malaria transmission and incidence in children aged 6 months to 10 years in the district of Koulikoro. Entomological parameters of malaria transmission during the last year of IRS implementation in 2016 were compared with those obtained 2 years after IRS withdrawal in 2018 in two villages of Koulikoro. Mosquito vectors were collected by mouth aspiration and pyrethrum spray catches in the villages to monitor these transmission parameters. A sharp increase (10.8 times higher) in vector abundance after IRS withdrawal was observed. The infection rate of Anopheles gambiae sensu lato to Plasmodium falciparum increased from zero during IRS implementation to 14.8% after IRS withdrawal. The average entomological inoculation rate, which was undetectable before, was 1.22 infected bites per person per month 2 years after IRS was withdrawn, and the cumulative malaria incidence rate observed after IRS was 4.12 times (15.2% versus 3.7%) higher than that observed in 2016 in the villages before IRS withdrawal. This study showed a resurgence of malaria transmission and incidence in the Koulikoro health district after IRS was withdrawn. Thus, to manage the potential consequences of malaria transmission resurgence, alternative approaches are needed when stopping successful malaria control interventions.