Highly pathogenic avian influenza A(H5N1) virus continues to expand globally across wild birds, poultry, and multiple mammalian hosts, and the detection of genetic features associated with mammalian adaptation underscores the need to better define preexisting human immunity relevant to pandemic preparedness. Although serologic and vaccine studies have historically emphasized hemagglutinin (HA), neuraminidase (NA) is a key antigen capable of providing protective immunity. Here, we performed a retrospective analysis of NA-specific antibodies in human sera collected across ten influenza seasons spanning the pre- and post-2009 pandemic periods. A total of 749 paired human serum samples (1,498 total) were obtained prior to seasonal inactivated influenza vaccination (T1) and 28 days after vaccination (T2). NA-inhibiting antibodies were measured by enzyme-linked lectin assay (ELLA) using N1 antigens representing three genotypes (B.3.13, D1.1, and EA-2021-AB) from clade 2.3.4.4b A(H5N1) viruses. In parallel, HA-directed responses were assessed by hemagglutination inhibition (HAI). In addition, luciferase-based microneutralization assays were performed to assess the presence of neutralizing antibodies. NA-inhibiting antibodies were detectable against all three genotypes across seasons. Seasonal vaccination was associated with a modest but reproducible increase in NA-inhibiting titers, most apparent from the 2009-2010 season onward, coincident with the introduction of A(H1N1)pdm09 into seasonal vaccine formulations. In contrast, HAI activity against H5 was generally low or undetectable, despite detectable HA-reactive antibodies by ELISA. These data indicate that cross-reactive NA-specific antibodies are present in human sera and can be boosted by seasonal vaccination, supporting increased consideration of NA in influenza serosurveillance and vaccine evaluation.