This study was performed to develop a new formula to estimate the renal and isthmus depth in horseshoe kidney, and to compare the new formula with previously published formulas.Renal depth, isthmus depth, vertebral thickness, and total thickness (T, cm) of the body at the level of the kidneys were measured by CT in 124 adults. Their sex, age, height (H, cm), and weight (W, kg) were recorded. Multiple stepwise linear regression analysis was conducted. The 124 cases were divided into 2 random groups, of which the first group was used to derive a regressive formula and the second group was used to verify the formula and compare the formula with previously published formulas.Multiple stepwise linear regression analysis showed that the important variables in estimating the depth of each kidney were the body weight (W, kg) and the total thickness (T, cm) of the body at the level of the kidneys. The important variables in estimating the depth of isthmus soft tissue and vertebral thickness were W, T, and age, W. The new formula was the following: right renal depth (cm) = 0.273 × T + 0.043 × W + 1.086 (r = 0.82, P < .05; standardized regressive coefficient: T = 0.500, W = 0.367), left renal depth (cm) = 0.245 × T + 0.041 × W + 0.676 (r = 0.83, P < .05; standardized regressive coefficient: T = 0.520, W = 0.353); isthmus depth (cm) = soft tissue depth + vertebral thickness, soft tissue depth (cm) = 0.144 × T + 0.044 × W + 0.536 (r = 0.58, P < .05; standardized regressive coefficient: T = 0.272, W = 0.335), vertebral thickness (cm) = 0.012 × age + 0.018 × W + 3.683 (r = 0.53, P < .05; standardized regressive coefficient: age = 0.326, W = 0.438). It is much better than the literatures.The new renal depth estimation formula in horseshoe kidney that we derived by using multiple stepwise linear regression has greatly outperformed other 6 previously published formulas. Isthmus depth estimation formula can also get accurate results. Our new formula provides a more reliable and accurate renal and isthmus depth estimation and contributes to improving the methods used to estimate renal function from radionuclide renography in horseshoe kidney.