Autotaxin (ATX) is a secreted phospholipase D, which cleaves lysophosphatidylcholin (LPC). ATXs product, lysophosphatidic acid (LPA), is an important cell motility and mitogenic factor which binds to its own group of receptors. ATX is upregulated in several cancer cell lines and has been shown to be a direct regulator of cancer growth and invasiveness. A DNA microarray study of adipose triglycerid lipase-knock out (ATGL-ko) mice showed differential regulation of ATX expression in various tissues. Furthermore, some ATGL-ko tumor cell-lines showed accelerated growth rates. To clarify if ATX expression and LPA production is interrelated to ATGL knock down and may be causative for increased cell proliferation of ATGL-ko cancer cell lines, we analyzed ATX expression and LPA levels in various ATGL-ko mouse tissues and several ATGL-ko cancer cell lines. ATX expression and activity levels in wild type and ATGL-ko Plasma and tissues were determined by qRT-PCR, Western blot and activity assays. For the measurement of LPA levels in tissue and plasma a derivatisation method for mass spectrometry was established. Our results show that ATX mRNA expression is differentially up- or down-regulated in various ATGL-ko mouse tissues. Interestingly, in plasma of ATGL-ko mice ATX activity was decreased as compared to that of control mice. Measurements of LPA in plasma of wild type and ATGL-ko mice showed slightly decreased levels of LPA. Notably, several ATGL-ko cancer cell lines showed after a recently performed ATGL Knock down significantly upregulated ATX expression. In summary, we found that ATX expression levels in various tissues and cells are affected by an ATGL-knock out or knock-down. The known changes in intracellular lipid signaling, following an ATGL knock down, could be suggested as explanation for this phenomenon.