Previous studies have found that obese people have lower levels of vitamin B, but most have focused on obesity as defined by body mass index (BMI), and its relationship with other types of obesity is unclear. The aim of this study was to explore the relationship between vitamin B levels and obesity assessed by different definitions among Chinese middle-aged and older community-dwelling adults. This cross-sectional study included 887 participants aged 45 years and older (45-82 years). The concentrations of vitamin B (B , B , B , and B ) were measured by robotic dry blood spot extraction systems in combination with liquid chromatography-tandem mass spectrometry. BMI, body fat percentage (BF%), visceral fat area (VFA), and waist circumference (WC) were used to diagnose obesity. VFA and BF% were assessed by bioelectrical impedance analysis. The logistic regression model was used to assess the associations between vitamin B levels and the odds of obesity. The average age of all participants was 60.77 (SD 6.33) years. The prevalence of obesity varied from 8.6% to 52.4% depending on different diagnostic criteria. After adjusting for covariates, a negative correlation was observed between vitamin B level and obesity according to the criteria of WC, VFA, and BF%, and the adjusted odds ratio (OR) was 0.47, 0.52, and 0.46, respectively. When using WC and BF% to define obesity, higher quartiles of vitamin B were negatively associated with the odds of obesity (OR: 0.62 and 0.62, respectively). Vitamin B was inversely associated with VFA-defined and BF%-defined obesity (OR: 0.64 and 0.64, respectively). When using VFA and BF% to define obesity, a negative correlation was observed in vitamin B (OR: 0.61 and 0.67, respectively). Vitamin B (B , B , B , and B ) level was negatively related to obesity (defined by WC, VFA, or BF%) in Chinese middle-aged and older adults.