The South African poultry industry has been beset by sporadic H6N2 avian influenza infection (sub-lineage I and II) in chickens since the early 2000s, with economic losses resulting from reduced egg production and co-infection with other pathogens. An egg-based inactivated H6N2 vaccine (AVIVAC® AI; Deltamune (Pty) Ltd.) based on a 2002 sub-lineage I isolate is available, although substantial antigenic drift has occurred in H6N2 viruses since its implementation. Globally, seasonal and pandemic plant-produced hemagglutinin (HA)- based influenza virus-like particle (VLP) vaccines are in advanced clinical trials with proven efficacy, speed of production, cost-effectiveness, scalability and safety, although not yet established for poultry. In this study, H6 avian influenza VLPs (sub-lineage I and II, respectively) were transiently produced in Nicotiana benthamiana and tested for protective efficacy in the target host. A production platform has been established for H6 VLPs in N. benthamiana by optimising protein expression and purification to maximize yield and by assessing the feasibility of large-scale production and downstream processing in a preliminary study. Subsequently, the respective plant-produced H6 VLPs were formulated into vaccines and their capacity to reduce viral replication and shedding upon challenge with a 2016 H6N2 field isolate were established in specific-pathogen-free (SPF) chickens, in comparison to the commercial H6N2 vaccine. The plant-produced sub-lineage I VLP vaccine (768 HA units/dose) was highly immunogenic (mean hemagglutination inhibition (HI) titer 10.7 log2), reduced the oropharyngeal and cloacal viral shedding by more than 100- and 6-fold, respectively, and shortened the duration of oropharyngeal shedding by at least a week in comparison to the non-vaccinated control. Due to initial low yield of sub-lineage II VLPs, the maximum antigenic mass vaccine dose (48 HA units/dose)) resulted in substantially lower HA-specific antibody titers (mean HI titer > 4 log2), but still reduced viral shedding from the oropharynx by more than 5-fold in comparison to the non-vaccinated control. In contrast, the commercial vaccine not only failed to effectively reduce shedding in comparison to the non-vaccinated control, but exacerbated oropharyngeal shedding until day 21 after viral challenge, illustrating the antigenic dissimilarity between the commercial vaccine and a recent field virus. Plant-produced VLP vaccines, which facilitates differentiation between infected and vaccination animals (DIVA), presents a new generation of poultry vaccines that is highly efficacious and cost-effective with the major advantage of producing a tailored antigenically-matched vaccine candidate within a short space of time and holds enormous potential for the poultry industry.