Vitamin D plays role in bone health and the regulation of the immune system. A cross-sectional study of serum 25-hydroxyvitamin D (25[OH]D) levels was conducted among HIV-1-infected Thai patients to determine the prevalence and associated factors of low vitamin D levels (25[OH]D <30 ng/mL) in tropical setting. 25-Hydroxyvitamin D was measured by liquid chromatography/tandem mass spectrometry. Of 178 patients, 58% received antiretroviral therapy at median (interquartile range [IQR]) duration of 7.4 (5.9-8.5) years. The prevalence of 25(OH)D deficiency (<20 ng/mL) and insufficiency (20-29.9 ng/mL) was 26.8% and 44.9%, respectively. Multivariate analysis showed that receiving efavirenz (EFV) was significantly associated with low vitamin D status (odds ratio = 3.60; 95% confidence interval, 1.06-12.15, P <.05). The mean (±standard deviation) level of 25(OH)D in patients receiving and not receiving EFV was 22.9 (6.6) and 28.6 (10.7) ng/mL, respectively, (P <.05). Low vitamin D status is common and needs to be assessed among HIV-infected patients including tropical residents especially when EFV is used.