Background: Several systemic factors and hormones are thought to regulate the fracture healing process. Vitamin D has emerged as a compound or hormone that actively participates in the regulation of calcium homeostasis and bone metabolism. Objectives: The aim of this study is to determine the serum changes in the level of vitamin D during the acute healing period of tibial and femoral shaft fractures. Patients and Methods: This cross-sectional study included of 73 patients with tibial and femoral shaft fractures referred to the Poursina Hospital between February 2011 and February 2012. Changes in the serum levels of vitamin D were assessed three times in a period of three weeks (at the first visit, end of first week, and end of the third week). Variables such as age, gender, fractured bone, concomitant fracture of tibia and fibula, type of fracture, time of measurement and serum levels of 25-hydroxyvitamin D were assessed. All statistical analyses were performed using the SPSS software. Results: Forty tibial fractures and 33 femoral fractures were recorded. Mean vitamin D levels at the time of admission, after one week and at the end of the third week for the 73 participants included in the study were 39.23, 31.49, and 28.57 ng/mL, respectively. The overall reduction of vitamin D level was significantly more evident in the first week versus the following (P < 0.0001). Conclusions: Serum levels of vitamin D in patients with tibial or femoral fractures were reduced during the curative period of the fracture. This can be related the role of vitamin D in the formation and mineralization of the callus. Patients with tibial or femoral shaft fractures may benefit from the administration of vitamin D supplements during the fracture healing process.