Preoperative radiotherapy (RT) for non-metastatic rectal cancer reduces local recurrence rates but can cause pelvic insufficiency fractures. Despite the high morbidity from RT-induced skeletal injuries, predictive and preventive measures are lacking. How these injuries are reflected by bone biomarkers are largely unknown. The aim was to assess longitudinal changes in bone biomarkers and their relation to RT-related bone injuries in women with rectal cancer. This longitudinal cohort study includes 47 women with non-metastatic rectal cancer treated with surgery +/- preoperative RT with or without chemotherapy. Sclerostin, bioactive sclerostin, C-terminal telopeptide cross-links of collagen type I (CTX), bone-specific alkaline phosphatase (BALP), and type I procollagen intact N-terminal propeptide (PINP) were measured at baseline, after RT, and 1 yr postoperatively. Pelvic magnetic resonance imaging was used for detection of skeletal injury. Sixteen of 36 (44%) irradiated women had radiation-induced bone injuries and were compared to 11 women (RT-) and 20 women (RT+) without bone injuries. Serum CTX, BALP, and PINP increased during the first year after RT in women with radiation-induced bone injuries. The difference in mean change of CTX (p=.037) and BALP (p=.042) was conferred by longitudinal regression analyses adjusted for serum estradiol. Serum sclerostin and bioactive sclerostin remained stable over time. Taken together, bone markers may be of interest for future research on fracture prediction or preventive measures in women susceptible to radiation-induced bone injury. Due to few measure points, the full pattern cannot be captured regarding the relation over time between bone biomarkers and skeletal injury from irradiation. Radiation therapy (RT) for rectal cancer before surgery reduces the risk of cancer recurrence, but can cause injuries to the pelvic bone. Despite the high morbidity from RT-caused skeletal injuries, predictive and preventive measures are lacking. How these bone injuries are reflected by bone biomarkers measured in serum is largely unknown. The aim of this study was to measure serum bone biomarkers and their relation to RT-related bone injuries in women with rectal cancer. The study group included 47 women with rectal cancer treated with surgery +/- preoperative RT. Pelvic magnetic resonance imaging was used for detection of skeletal injury. The bone biomarkers sclerostin, bioactive sclerostin, C-terminal telopeptide cross-links of collagen type I (CTX), bone-specific alkaline phosphatase (BALP), and type I procollagen intact N-terminal propeptide (PINP) were measured at baseline, after RT, and 1 yr after surgery. Sixteen of 36 (44%) irradiated women had radiation-caused bone injuries and were compared to 31 women without bone injuries. Serum CTX, BALP, and PINP increased during the first year after RT in women with radiation-caused bone injuries. In summary, bone markers may be of interest for future research on fracture prediction or preventive measures in women prone to radiation-caused bone injury.