Mycoplasma bovis is a global concern for the cattle industry owing to its high rates of infection and resulting morbidity, but there are no reports on the lipid composition and metabolic pathways. This study examined the lipidomics of M. bovis to better investigate the biological characteristics of clinical isolates of M. bovis. The M. bovis strains NX114 and PG45, cultivated to log-phase, underwent untargeted lipidomics via ultra-high-performance liquid chromatography-mass spectrometry for differential analysis. Over 65 lipid types and 1476 lipid molecules were identified. We found that glycerophospholipids and sphingolipids constitute the predominant lipid components of M. bovis, with significant constituents of its cell membrane comprising ceramides, phosphatidylglycerols, sphingomyelins, triacylglycerols, hexosylceramides, ether-linked oxidized phosphatidylcholines, and oxidized phosphatidylglycerols. Secondly, 562 differentially expressed lipid molecules were identified, including 17 lipid subclasses (15 up-regulated and 2 down-regulated) with significant differences in relative content. These findings indicate that distinct M. bovis isolates contain structurally varied lipid molecules, with sphingomyelin, phosphatidylinositol, cardiolipin, and phosphatidylcholine being characteristic lipids. The glycerophospholipid metabolism pathway was identified as a key pathway regulating lipid metabolism in M. bovis by KEGG pathway enrichment analysis. The results indicated alterations in the lipid metabolism of M. bovis, offering insights into its pathogenic mechanisms.