Objectives: Clinical guidelines disagree on the diagnostic usefulness of Borrelia burgdorferi (Bb) serum antibodies (serum-Bb) in investigation of Lyme neuroborreliosis (LNB). We investigated the association between serum-Bb and Bb intrathecal antibody index (Bb-AI) and rates of seroconversion and seroreversion after LNB. Methods: Danish residents who had a Bb-AI and corresponding serum-Bb measured between 1994 and 2020 were identified at all Danish departments of clinical microbiology. We used descriptive statistics to examine the proportions of positive Bb-AI combined with positive or negative serum-Bb antibody tests. Next, the rate of seroconversion and seroreversion among those with positive Bb-AI and either an initial negative or positive serum-Bb was estimated. Results: We included 34 609 individuals with a Bb-AI and corresponding serum-Bb. The proportion of individuals with positive Bb-AI who had negative serum-Bb was 16.8% (95% CI, 15.1–18.6). The proportion of individuals with positive serum-Bb IgM, serum-Bb IgG, or serum-Bb IgM and IgG antibodies who had positive Bb-AI was 10.6% (95% CI, 9.5–11.8), 24.7% (95% CI, 23.0–26.4), and 45.0% (95% CI, 42.4–48.0), respectively. The proportion of children (<18 years) with positive serum-Bb IgM and IgG antibodies who had a positive Bb-AI was 59.7% (95% CI, 53.4–65.8). The proportion of individuals with positive Bb-AI with initial negative or positive serum-Bb antibodies who seroconverted or seroreverted within 2 years was 17.3% (95% CI, 6.9–27.8) and 23.2% (95% CI, 19.1–27.7), respectively. Conclusions: Serum-Bb antibodies could not predict results of Bb-AI. A fifth of both seronegative and seropositive individuals with positive Bb-AI seroconverted or seroreverted within 2 years.