Calcium (Ca) is required for the growth and development of sheep, but the requirement of Yunnan semi-fine wool (YSW) rams remains uncovered. The current study aims to estimate the Ca requirement of growing YSW rams based on their growth performance, Ca utilization, and serum biochemical indexes. Forty-five YSW rams (10-month-olds) were randomly allocated to five dietary treatments with varying Ca levels of 0.50% (D1), 0.68% (D2), 0.73% (D3), 0.89% (D4), and 0.98% (D5). A higher value for average daily gain and a lower value for the feed conversion ratio were observed in the D3 group compared to the D5 group (p < 0.05). The dry matter intake amount changed quadratically with the increased Ca levels (p < 0.05). The levels of Ca intake, fecal Ca, and excreted Ca were significantly higher in the D5 group than those in the D1 group (p < 0.05). The apparent Ca digestibility rate and the Ca retention rate were significantly higher in the D4 group than in the D1 group (p < 0.05). The serum Ca concentration increased linearly with the incremental levels of dietary Ca (p < 0.05). The activity of alkaline phosphatase was significantly higher in the D1 group than in the D2 group (p < 0.05). The serum levels of hydroxyproline, osteocalcin, and calcitonin decreased from the D1 group to the D2 group, and then significantly ascended (p < 0.05) with the dietary Ca levels from the D3 group to the D5 group. The serum parathyroid hormone content was elevated from the D1 group to the D3 group and then decreased from the D4 group to the D5 group. After calculation, the daily net Ca requirement for the maintenance of YSW rams was 0.073 g/kg of BW0.75, and the daily total Ca requirement was 0.676 g/kg of BW0.75. To optimize the growth performance and the Ca utilization of YSW rams, the recommended dietary Ca level ranges from 0.73% to 0.89% based on this study.