Staphylococcus aureus is a Gram-positive opportunistic human pathogen that can cause severe infection such as endocarditis, sepsis, and toxic shock syndrome. Due to rising antibiotic resistance, as seen for example in Methicillin-resistant S. aureus stains (MRSA), treatment of infection becomes more and more challenging, making S. aureus to one of the most important pathogens in healthcare. In the late 1990s, it was discovered that S. aureus can reside within non-professional phagocytic cells (NPPC), such as endothelial and epithelial cells, a feature that often was associated with antibiotic resistance as well as chronic infection. Thus, we try to understand how S. aureus is able to enter its host cells and which strategies are used by the pathogen to replicate within the intracellular niche and moreover, how these processes could be targeted for treating S. aureus infection.Here, we provide evidence that the uptake of the MRSA strain JE2 by host cells requires liberation of Ca2+ from the endolysosomal compartment and activity of acid sphingomyelinase (ASM), a lysosomal enzyme involved in the degradation of the sphingolipid sphingomyelin. ASM likely remodels plasma membrane during pathogen internalization resulting in a rapid uptake, whereas other invasion pathways that are ASM-independent possess slower kinetics. Furthermore, we established an APEX2-based proximity labeling approach that enables identification of host molecules that interact with S. aureus. Thereby, we found novel receptor candidates that likely are enrolled in the ASM-dependent internalization pathway.In NPPCs, S. aureus lyses the phagosomal membrane and invades the cytosol of host cell, a process that we call phagosomal escape. We found that phagosomal escape rates of bacteria entered their host cell via the ASM-dependent pathway are lower than for bacteria that used other internalization pathways, suggesting that the outcome of an intracellular S. aureus infection is at least partially decided at the host"s plasma membrane. Moreover, we found that pharmacological inhibition of ASM protected host cells from infection, suggesting ASM as a promising target for treatment of S. aureus infection.