Sphingosine 1-phosphate (S1P), a bioactive lipid, interacts with five widely expressed G proteincoupled receptors (S1P1-5), regulating a variety of downstream signaling pathways with overlapping but also opposing functions. To date, data regarding the role of S1P5 in cell proliferation are ambiguous, and its role in controlling the growth of untransformed cells remains to be fully elucidated. In this study, we examined the effects of S1P5 deficiency on mouse embryonic fibroblasts (MEFs). Our results indicate that lack of S1P5 expression profoundly affects cell morphology and proliferation. First, S1P5 deficiency reduces cellular senescence and promotes MEF immortalization. Second, it decreases cell size and leads to cell elongation, which is accompanied by decreased cell spreading and migration. Third, it increases proliferation rate, a phenotype rescued by the reintroduction of exogenous S1P5. Mechanistically, the anti-proliferative function of the S1P/S1P5 axis is associated with reduction in nuclear accumulation of activated ERK. Our results suggest that S1P5 opposes the growth-promoting function of S1P1-4 through spatial control of ERK activation and provide new insights into the anti-proliferative function of S1P5.