Long-chain n-3 PUFA (LCPUFA) and palmitate (16:0) positioning in the triacylglycerol (TAG) of infant formula may affect calcium-uptake which could affect bone health. We investigated if a human milk fat substitute (HMFS) with a modified TAG structure holding 16:0 predominantly in the sn-2-position compared with a control (CONT) and if increasing n-3LCPUFA intake giving fish oil (FO) compared with sunflower oil (SO) would affect bone parameters in piglets in two sets of controlled 14d-interventions (n=12/group). We assessed this by dual-energy x-ray absorptiometry, and ex vivo peripheral quantitative computed tomography and mechanical strength. Bone mineral content (BMC) was higher in the FO compared to the SO-group (p=0.03). Despite similar weight gain in HMFS- and CONT-groups, body fat accumulation was higher with HMFS (p<0.001), and BMC, bone area (BA) and cortical BA in femur were lower (p=0.002, p=0.005, and p=0.02, respectively), indicating importance of both n-3LCPUFA and 16:0 TAG-positioning in infant formulas.