Low pathogenic avian influenza viruses (LPAIVs) are typically associated with subclinical or mild disease in poultry. However, recent outbreaks involving atypical LPAIV strains, including H3N1 strains like A/chicken/Belgium/460/2019, have demonstrated severe clinical outcomes despite low intravenous pathogenicity index (IVPI) scores. These findings challenge current classification systems and raise questions about alternative markers of virulence, such as loss of a neuraminidase (NA) glycosylation site linked to plasminogen-binding and haemagglutinin (HA) cleavage. This study compared the pathogenicity of a wild-type H3N1 strain (wtH3N1), isolated from a disease outbreak in Belgium, with a genetically modified, loss-of function variant (mH3N1) carrying a single amino acid substitution (S122N) in NA that blocks plasminogen-dependent cleavage of HA in chickens. Four-week-old pullets and cockerels, and 30-week-old laying hens were inoculated with either wtH3N1 or mH3N1 and clinical signs, egg production, viral replication, post-mortem and tissue pathology were evaluated. Adult hens infected with wtH3N1 showed a complete cessation of egg production, systemic viral replication, and histopathological lesions in the reproductive tract, brain, and kidneys. In contrast, birds infected with mH3N1 displayed only mild, transient reductions in egg production and minimal viral detection. No mortality was observed in any group. All young chickens exhibited subclinical infections. Overall, the S122N mutation significantly attenuated viral virulence and tissue tropism. The study provides functional evidence that position 122 on NA contributes to increased virulence in H3N1 AIV. These findings support the role of molecular markers in risk assessment of non-H5/H7 LPAIVs and highlight the limitations of the current IVPI-based classification system.