Purpose Low vitamin D levels have been associated with musculoskeletal pain, cancer pain, chronic postoperative pain, and post-traumatic pain. However, their association with postoperative pain after video-assisted thoracoscopic surgery has not been explored. The aim of this study was to examine the association between vitamin D levels and postoperative pain after video-assisted thoracoscopic surgery. Patients and Methods This study enrolled 194 adult patients who underwent elective non-cardiac thoracic surgery in Shanghai Pulmonary Hospital from February 2021 to June 2021. Following application of the exclusion criteria, 135 patients who underwent video-assisted thoracoscopic surgery were included in the final analysis. The primary outcome was the incidence of acute postoperative moderate-severe pain. Secondary outcomes included C-reactive protein (CRP), interleukin (IL)-1, IL-6, and tumor necrosis factor-α levels in the immediate postoperative (48 hours) period, as well as pain scores at 3 months after surgery. A multivariable logistic regression model was used to analyze the association between vitamin D levels and acute postoperative moderate-severe pain. Results Among 135 patients, 54.1% were categorized as having a low vitamin D level (<30 nmol/L). On multivariable analysis, patients with a low 25-hydroxy-vitamin D (25[OH]D) level had a higher risk of postoperative moderate-severe pain (odds ratio, 2.44; 95% confidence interval, 1.181–5.041; P = 0.016) when compared to patients with a sufficient 25(OH)D level. Static and dynamic pain scores at 3 months after surgery, as well as serum levels of CRP, IL-1, IL-6, and tumor necrosis factor-α were not significantly different between patients with low and sufficient 25(OH)D levels. Conclusion Patients with low vitamin D levels are at a higher risk of acute moderate-severe pain after video-assisted thoracoscopic surgery. Trial Registration http://www.chictr.org.cn , ChiCTR2100052380.