Sphingosine-1-phosphate (S1P), a sphingolipid with second messenger properties, is a main regulator of various cellular processes including lymphocyte cell trafficking, angiogenesis, cell proliferation, and survival. High S1P concentrations and deficiencies in S1P degradation have been associated with cancer cell progression, their directed chemoattraction and promotion of chemo-resistance mechanism. The endoplasmic reticulum (ER) membrane localized enzyme sphingosine-1-phosphate lyase (SGPL1) has a key role in prevention of S1P overstimulation in tumor cells by its irreversible S1P degradation activity. In this paper we demonstrated a SGPL1 overexpression and mislocalization in pediatric alveolar rhabdomyosarcoma (RMA) cells. Moreover, a homozygous point mutation from A to G at position 321 in the coding sequence was obvious, which interferes with the S1P degradation activity and correct localization in the ER-membrane. By complementation with the native SGPL1 variant, the ER localization was restored in RMA cells. More importantly, the SGPL1 restauration prevents the S1P induced migration and colony formation of RMA cells, significantly. This observation opens new highways for the treatment of pediatric RMA by gene therapeutic SGPL1 renewal and recommends the detection of specific SGPL1 mutations as pathological, molecular metastasis marker.