Sphingolipids are essential biomolecules and membrane components, but their regulatory role in cotton fiber development is poorly understood. Here, we found that fumonisin B1 (FB1)-a sphingolipid synthesis inhibitor-could block fiber elongation severely. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we detected 95 sphingolipids that were altered by FB1 treatment; of these, 29 (mainly simple sphingolipids) were significantly increased, while 33 (mostly complex sphingolipids) were significantly decreased. A quantitative analysis of the global proteome, using an integrated quantitative approach with tandem mass tag (TMT) labeling and LC-MS/MS, indicated the upregulation of 633 and the downregulation of 672 proteins after FB1 treatment. Most differentially expressed proteins (DEPs) were involved in processes related to phenylpropanoid and flavonoid biosynthesis. In addition, up to 20 peroxidases (POD) were found to be upregulated, and POD activity was also increased by the inhibitor. To our knowledge, this is the first report on the effects of FB1 treatment on cotton fiber and ovule sphingolipidomics and proteomics. Our findings provide target metabolites and biological pathways for cotton fiber improvement.