Roux-en-Y gastric bypass may lead to impaired calcium uptake. Therefore, operation-specific effects of gastric bypass and vertical banded gastroplasty on bone mineral density (BMD) were examined in a randomized clinical trial. Bone resorption markers and mechanisms of decreased calcium uptake after gastric bypass were investigated using blood and endoscopic samples from two additional patient cohorts. Total BMD and non-weight-bearing skull BMD were measured by dual-energy X-ray absorptiometry at baseline, and 1 and 6 years after gastric bypass or vertical banded gastroplasty in patients who were not receiving calcium supplements. Bone resorption markers in serum and calcium uptake mechanisms in jejunal mucosa biopsies were analysed after gastric bypass by proteomics including radioimmunoassay, gel electrophoresis and mass spectrometry. One year after surgery, weight loss was similar after gastric bypass and vertical banded gastroplasty. There was a moderate decrease in skull BMD after gastric bypass, but not after vertical banded gastroplasty (P < 0·001). Between 1 and 6 years after gastric bypass, skull BMD and total BMD continued to decrease (P = 0·001). C-terminal telopeptide levels in serum had increased twofold by 18 months after gastric bypass. Proteomic analysis of the jejunal mucosa revealed decreased levels of heat-shock protein 90β, a co-activator of the vitamin D receptor, after gastric bypass. Despite increased vitamin D receptor levels, expression of the vitamin D receptor-regulated calcium transporter protein TRPV6 decreased. BMD decreases independently of weight after gastric bypass. Bone loss might be attributed to impaired calcium absorption caused by decreased activation of vitamin D-dependent calcium absorption mechanisms mediated by heat-shock protein 90β and TRPV6.