The rapid increase in bone turnover occurring when discontinuing long-term treatment with denosumab (DMAB), an antibody that neutralizes receptor activator of nuclear factor kappa-B ligand (RANKL) is not fully understood. We aimed to investigate the mechanisms underlying the rebound activation of bone resorption by measuring tartrate-resistant acid phosphatase 5b (TRAcP 5b), RANKL, osteoprotegerin (OPG), C-terminal collagen crosslinks (CTX), and procollagen type I N-propeptide (P1NP) in patients discontinuing long-term DMAB. Sixty-one patients with BMD T-score > - 2.5 at the spine and hip discontinuing long-term DMAB were randomized to treatment with zoledronate (ZOL) 6months (6M group, n = 20), 9months (9M group, n = 20) or 12months after the last DMAB injection or when bone turnover was high (12M group, n = 21). Bone turnover markers were measured immediately before initiation of ZOL treatment. We found higher CTX and PINP in the 9M and 12M groups compared to the 6M group (p < 0.001). In the 6M group, TRAcP 5b was lower and RANKL higher than in the other two groups (p < 0.001). TRAcP 5b correlated negatively with RANKL (R = - 0.54), and time since the last DMAB injection correlated positively with CTX (R = 0.56), PINP (R = 0.72), TRAcP 5b (R = 0.51) and negatively with RANKL (R = - 0.70) (p < 0.001 for all). We found no difference in OPG between groups. Following discontinuation of long-term DMAB, we find high levels of RANKL, which most likely result in an increase in the number of active osteoclasts (illustrated by TRAcP5b) causing an increased bone turnover.