We demonstrated murine leukemia inhibitory factor (mLIF) mRNA in osteoblastic MC3T3-E1 cells, but not mLIF in their conditioned medium. Recombinant mLIF had an inhibitory effect on alkaline phosphatase (ALP) activity, but not on DNA synthesis, in these mLIF-free cells. This inhibitory effect was not prostaglandin E2 dependent. mLIF also modulated the inhibitory effect of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] on ALP activity, partly via down regulation of 1,25(OH)2D3 binding sites. These results suggest that LIF may play a role in regulating osteoblast differentiation.