Background <p>Insecticide-treated nets (ITNs) incorporating pyrethroids with piperonyl butoxide (PBO) have demonstrated enhanced effectiveness against malaria transmitted by pyrethroid-resistant vectors compared with standard pyrethroid-only ITNs. To sustain progress in malaria prevention, a broader portfolio of effective pyrethroid-PBO nets is required to promote market diversity and strengthen supply resilience. In this study, we evaluated the entomological efficacy and wash durability of Vector Guard®, a novel mosaic alpha-cypermethrin–PBO ITN, against pyrethroid-resistant <i>Anopheles gambiae</i> sensu lato (<i>An. gambiae</i> s.l.) in southern Benin.</p> Methods <p>An experimental hut trial was conducted in Covè, Benin, against wild, free-flying <i>An. gambiae</i> s.l. The effectiveness of the Vector Guard® ITN was tested unwashed and after 20 standardized washes, and also compared to two WHO-prequalified ITNs: Olyset® Plus (a permethrin-PBO net) and Royal Sentry® 2.0 (an alpha-cypermethrin-only net). Primary outcomes were mosquito mortality and blood-feeding protection. Susceptibility bioassays were conducted to assess local vector resistance to insecticides. Laboratory cone and tunnel tests were also performed to help explain the finding in the experimental huts. Chemical content analyses were performed to investigate active ingredient wash retention. Vector Guard® was assessed for its non-inferiority to Olyset® Plus following WHO guidance.</p> Results <p>The wild <i>An. gambiae</i> s.l. population at Covè exhibited high frequencies of pyrethroid resistance, with PBO pre-exposure restoring partial susceptibility to alpha-cypermethrin (34% vs 4% mortality) but not to permethrin (2.0% vs 2.1% mortality). A total of 6799 females were collected in the experimental huts. Vector Guard® outperformed both Royal Sentry® 2.0 and Olyset® Plus across all entomological endpoints. Mortality with Vector Guard® was significantly higher than with Olyset® Plus both when unwashed (36.4% vs 17.5%, <i>p</i> &lt; 0.001) and after 20 washes (17.2% vs 8.7%, <i>p</i> &lt; 0.001). Non-inferiority analysis with pooled data for unwashed and washed nets confirmed that Vector Guard® was non-inferior to Olyset® Plus in terms of both mortality (odds ratio [OR] 2.71, 95% confidence interval [CI] 2.26–3.24, non-inferiority margin [NIM] 0.423) and blood-feeding protection (OR 0.53, 95% CI 0.45–0.62, NIM: 1.359). These findings were supported by the results from the cone and tunnel tests. Chemical analysis showed higher wash retention of active ingredients in Vector Guard® (83% for PBO and &gt; 94% for alpha-cypermethrin) compared to Olyset® Plus (40.2% for PBO and 69.6% for permethrin).</p> Conclusions <p>Vector Guard® demonstrated superior entomological efficacy and wash durability compared to Royal Sentry® 2.0 and Olyset® Plus, and fulfilled WHO non-inferiority criteria for mosquito mortality and blood-feeding inhibition. These findings support its addition to the WHO list of prequalified pyrethroid-PBO ITNs and its potential to provide improved malaria control when deployed on a large scale in areas with high levels of pyrethroid resistance.</p> Graphical Abstract <p></p>