To modify the structure of milk fat globules (MFGs), raw milk (RM) with native MFGs, and milk with small (MS) and large (ML) MFGs, were subjected to moderate pulsed electric field (PEF) treatment at 9 and 16 kV·cm⁻¹ for 30 μs. The changes caused by PEF on MFG structure leading to MFG-MFG and MFG-protein aggregation were found to depend on the size, composition and/or previous damage caused by the separation and mixing processes during production of MS and ML, in addition to the intensity of the PEF treatment. Although, PEF treatment had a more pronounced effect on MFGM proteins of MS, these MFG were less affected by PEF due to significant adsorption of milk proteins. The structural changes caused by PEF were accompanied by changes in lipase activity and fatty acid (FA) profiles, i.e. increase in the proportion of C6:0, C8:0, C10 and some long-chain FAs and a decrease in medium and other long chain FAs. Industrial relevance: PEF is gaining attention in the dairy industry. However, information regarding the effect of PEF processing on milk components and functionality is still limited. The present investigation contributes to understand the potential of PEF at moderate field intensities to modify the MFGs and consequences for milk stability, lipolysis and composition of FAs. The new insight is relevant in development of new products and ingredients containing milk fat.