The role of sphingolipids in bacterial pathogenesis has been gradually recognized. In an effort to identify the possible involvement of sphingolipids during Mycoplasma pneumoniae (M. pneumoniae) infection, we first adopted a lipidomic approach to achieve the profiles of major sphingolipid species of M. pneumoniae as well as human lung carcinoma A549 cells, and further evaluated the effects of M. pneumoniae infection on sphingolipid metabolism in A549 cells. It was shown that M. pneumoniae and A549 cells share many common sphingolipid species, however, M. pneumoniae possesses certain specific molecular species that are not found in A549 cells. On the other hand, M. pneumoniae infection could alter sphingolipid metabolism in A549 cell, including the generation of new ceramide and sphingomyelin species, or the increase/decrease of intensities, which varies depending on the different infection doses and times. The effects of M. pneumoniae infection on two key enzymes in sphingolipid metabolism, serine palmitoyltransferase (SPT) and acid sphingomyelinase (ASM), were also examined. It was found that M. pneumoniae infection could affect the expression of SPT or the distribution of ASM at certain concentrations. These data suggest that M. pneumoniae infection could influence sphingolipid metabolism of its host, which might be related to its pathogenicity.