To investigate the effects of a critical enzyme, cerebroside sulfotransferase (CST), involving sulfatide biosynthesis on lipid (particularly sphingolipid) homeostasis, herein, we determined the lipidomes of brain cortex and spinal cord from CST null and heterozygous (CST(-/-) and CST(+/-), respectively) mice in comparison to their wild-type littermates by multi-dimensional mass spectrometry-based shotgun lipidomics. As anticipated, we demonstrated the absence of sulfatide in the tissues from CST(-/-) mice and found that significant reduction of sulfatide mass levels was also present, but in an age-dependent manner, in CST(+/-) mice. Unexpectedly, we revealed that the profiles of sulfatide species in CST(+/-) mice were significantly different from that of littermate controls with an increase in the composition of species containing saturated and hydroxylated fatty acyl chains. Contrary to the changes of sulfatide levels, shotgun lipidomics analysis did not detect significant changes of the mass levels of other lipid classes examined. Taken together, shotgun lipidomics analysis demonstrated anticipated sulfatide mass deficiency in CST defect mouse brain and revealed novel brain lipidome homeostasis in these mice. These results might provide new insights into the role of CST in myelin function.