In this study, the effects of different microwave treatment times (10, 20, 30, 40, 50, and 60 s) on fat loss, fatty acid profile, lipid transfers, and microstructure of pork subcutaneous back fat (PSBF) were investigated. Results showed that the fat loss of PSBF after microwave treatment for 60 s was significantly increased (53.31%). Microwave treatment increased the proportion of saturated fatty acids but reduced the relative content of monounsaturated and polyunsaturated fatty acids. Cryo-scanning electron microscopy, ordinary optical microscope and confocal laser scanning microscope analysis demonstrated that microwave treatment facilitated a large outflow of fat from the adipocytes, and the increasing and then decreasing changes in the size of individual adipocytes. Magnetic resonance imaging enabled the visualization of fat migration of PSBF. Overall, PSBF can be used as a biological model to evaluate the effect of microwave treatment on the change and migration mechanism of fat.