The aim of the study is to determine whether serum 25-hydroxyvitamin D (25(OH)D) deficiency in infants increased odds of urinary tract infection (UTI). A total of 238 infants including 132 patients experiencing a first episode of UTI and 106 controls, aged from 1 to 12 months, were enrolled. Serum 25(OH)D levels were tested through blood sampling. The serum 25(OH)D levels were significantly lower in cases with UTI than controls. The mean serum 25(OH)D levels were 29.09±9.56 ng/mL in UTIs and 38.59±12.41 ng/mL in controls (P<0.001). Infants with acute pyelonephritis (APN) had lower serum 25(OH)D than those with lower UTI. The multivariate logistic regression analyses showed that serum 25(OH)D<20 ng/mL (OR 5.619, 95% CI 1.469–21.484, P=0.012) was positively related to an increased odds of UTI. Vitamin D supplementation (OR 0.298, 95% CI 0.150–0.591; P=0.001) was associated with a decreased likelihood of UTI. Vitamin D deficiency in infants was associated with an increased odds of UTI. Interventional studies evaluating the role of vitamin D supplementation to reduce the burden of UTI are warranted. Abbreviations: 25(OH)D = 25-hydroxyvitamin D, APN = acute pyelonephritis, CAKUT = congenital anomalies of the kidney and urinary tract, CFU = colony forming unit, CI = confidence interval, CRP = C-reactive protein, DMSA = dimercaptosuccinic acid, IOM = Institute for Organization Management, IU = international unit, OR = odds ratio, SD = standard deviation, SPSS = statistical package for the social sciences, UTI = urinary tract infection, VCUG = voiding cystourethrography, VUR = vesicoureteral reflux, WBC = white blood cell.