Background: Osteoporosis and obesity are global health problems. Milk is high in n-3 alpha-linolenic acid (ALA), conjugated linoleic acid (CLA), and calcium, all of which are regarded as health beneficial by promoting bone formation and decreasing adiposity. This study examined the interaction among these milk components and the mechanisms underlying this regulation. Methods: Mouse ST2 stromal, MC3T3-L1 adipocyte-like, and MC3T3-E1 osteoblast-like cells were treated with: 1) ALA with LA:ALA=1-5:1; 2) individual/combinations of 20 µM cis-9,trans-11 (9,11) and trans-10,cis-12 (10,12) CLA isomers (80:10, 90:10, or 90:5%); 3) calcium phosphate (0.5-3.0 mM); or 4) combinations of ALA, CLAs, and calcium, with a slight modification, accordingly, during proliferation (8 days) and adipogenic and/or osteoblastic differentiation (6 days). Following the oil red O and alizarin red S staining, quantification of triglyceride accumulation and calcium deposition was performed. Secretion of eicosanoids and growth factors was determined from differentiation media. Results: ALA with LA:ALA=1-5:1 constantly inhibited proliferation/differentiation of MC3T3-L1 but facilitated MC3T3-E1 cell differentiation, showing maximal osteoblastogenesis and minimal adipogenesis at LA:ALA=4:1. At this level, insulin-like growth factor-1 (IGF-1) and IGF binding protein-3 (IGFBP-3) production was lowest in MC3T3-L1 cells, implying that ALA may regulate adipocyte differentiation via IGF-1/IGFBP-3 signaling pathway. Various combinations of 9,11/10,12-CLA mixtures had a tendency to inhibit MC3T3-L1 and MC3T3-E1 cell proliferation. During differentiation, combined 9,11-/10,12-CLAs, unlike individual isomers having a negligible effect on both cell growth, exerted a promising outcome by further decreasing adipocytic and increasing osteoblastic differentiation. In both cells, most of CLA isomer mixtures resulted in increased (but not significant) production of prostaglandin E2 (PGE2). The 1.5-2.5 mM calcium level was the best by promoting ST2 and MC3T3-E1and inhibiting MC3T3-L1 cell proliferation. Incorporation of ALA, CLA isomers, and calcium generally decreased ST2 and MC3T3-E1 but not MC3T3-L1 cell proliferation. During differentiation, however, ALA (4:1)+CLA (90:10%)+calcium (2.0 mM) significantly attenuated lipid accumulation in MC3T3-L1 and increased calcium deposition in MC3T3-E1 cells, in which PGE2 and leukotriene B4 (LTB4) production was increased in MC3T3-L1, whereas IGF-1 secretion was decreased in MC3T3-E1 cells, implying the possible benefit of this dietary regimen in promoting bone health by facilitating bone formation and reducing adiposity. Conclusions: These findings suggest that a diet with LA:ALA=4:1 is optimal to improve bone health, which can be further enhanced when incorporated with CLA (9,11:10,12=90:10%) and high calcium (2.0 mM).