We have identified a mechanism to diminish proliferation capacity of cells during cell expansion by using human adipose―derived stromal cells (hAD―SCs) as a model of replicative senescence. hAD―SCs of high passage numbers exhibited a reduced proliferation capacity with accelerated cellular senescence. The levels of key bioactive sphingolipids were significantly increased in these senescent hAD―SCs. Notably, transcription of sphingosine kinase 1 (SPHK1) was down-regulated in hAD―SCs at high passage numbers. SPHK1 knockdown as well as inhibition of its enzymatic activity impeded proliferation of hAD―SCs, with concomitant induction of cellular senescence and accumulation of sphingolipids as in high passage cells. SPHK1 knockdown―accelerated cellular senescence was attenuated by co―treatment of sphingosine―1―phosphate and an inhibitor of ceramide synthesis, fumonisin B1 but not by treatment of either one. Together, these results suggest that down―regulated transcription of SPHK1 is a critical inducer of altered sphingolipid profiles, enhancing replicative senescence during multiple rounds of cell division.