BackgroundHearing loss (HL) is increasingly recognized as a significant global public health issue, and research on its relationship with vitamin D levels has gained wider attention. However, the association between serum biomarkers 25-hydroxyvitamin D2 (25(OH)D2) and D3 (25(OH)D3) with different types of HL remains unclear. This study aimed to investigate the potential association of serum 25(OH)D2 and 25(OH)D3 with HL in US adults.MethodsA sample of 3,684 individuals aged 20–69 years from the 2015–2016 National Health and Nutrition Examination (NHANES) was analyzed in this study. HL was defined as a pure tone average > 25 dB in either ear at low frequencies (500, 1,000, 2000 Hz), speech frequencies (500, 1,000, 2000, 4,000 Hz), and high frequencies (3,000, 4,000, 6,000, 8,000 Hz). Logistic regression was employed to examine the association between serum 25(OH)D2 and 25(OH)D3 and HL. The study population was then stratified by age, gender, race, and education level to analyze potential differences between adults in different subgroups.ResultsIn the multivariate analysis, it was found that serum 25(OH)D2 was independently associated with low-frequency hearing loss (LFHL) (OR: 1.012 [95% CI, 1.005–1.020]) and speech-frequency hearing loss (SFHL) (OR: 1.011 [95% CI, 1.003–1.018]). Restrictive cubic spline analysis demonstrated a linear dose–response relationship between serum 25(OH)D2 levels and LFHL (p for linearity <0.001), as well as SFHL (p for linearity = 0.001). Conversely, an L-shaped association was observed between serum 25(OH)D3 levels and both LFHL (p for nonlinearity = 0.014) and SFHL (p for nonlinearity = 0.025), with threshold values identified at 35.3 and 36.5 nmol/L, respectively. Higher levels of serum 25(OH)D3 were associated with a lower probability of high-frequency hearing loss (HFHL) (OR: 0.994 [95% CI, 0.989–0.999]), with a threshold value identified at 53.9 nmol/L. Furthermore, a significant interaction between diabetes and serum 25(OH)D2 in LFHL was revealed through subgroup analysis (p = 0.041). In the non-diabetic population, serum 25(OH)D2 maintained its association with LFHL.ConclusionOur findings suggested a positive association between serum 25(OH)D2 concentrations and both LFHL and SFHL in the studied cohort. Additionally, an L-shaped relationship was found between serum 25(OH)D3 and LFHL and SFHL, and higher levels of serum 25(OH)D3 were identified to be associated with a lower risk of HFHL.