Arp2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including actin-related proteins (Arps) 2 and 3. In isolation Arp2/3 complex is in an inactive, splayed conformation. Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions and contribution of each NPF to activation are unresolved. Here, we report the cryo-EM structure of recombinantly-expressed human Arp2/3 complex with two WASP-family NPFs bound and address the mechanism of activation. A crosslinking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation, but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex. In addition to the complex being activated by NPFs, the complex is negatively regulated by a homologous protein, Arpin. Here, we also report the cryo-EM structure of bovine Arp2/3 complex bound to human Arpin. We’ve shown that despite its sequence similarity to the NPFs, Arpin specifically binds to a single binding site on Arp3. We have also shown that unlike GMF, Arpin acts on both ADP and ATP Arp2/3 complex. Here we have identified a unique mechanism of inhibition on Arp2/3 complex by Arpin.