Electrospun membranes are widely used in tissue engineering. Regretfully, there is limited research on how its morphological characteristics precisely regulate macrophage activation and immune response. Therefore, electrospun poly-l-lactic acid (PLLA) membranes with different alignments (align and random) and diameters (nanoscale and microscale) are prepared to investigate the effects of different surface morphologies on M2 macrophage polarization. Additionally, transcriptome, proteome, and phosphoproteome sequencings are combined to examine the underlying regulatory mechanisms. The results show that the electrospun PLLA membranes with different surface morphologies have good biocompatibility and can regulate the phenotype and function of macrophages by changing the micromorphology of the matrix surface. Especially, macrophages cultured on the electrospun membranes of the A600 group exhibit higher M2 macrophage polarization than the other three groups. Furthermore, the findings demonstrate that electrospun PLLA membranes enhance AMP-activated protein kinase (AMPK)/ mammalian target of rapamycin (mTOR) signaling activation by upregulating the expression of integrin phosphoenolpyruvate carboxykinase 2 (PCK2), which is critical for M2 macrophage polarization. Taken together, electrospun PLLA membranes promote M2 macrophage polarization by regulating the PCK2/AMPK/mTOR signaling pathway. This research can provide further theoretical bases for scaffold design, immunoregulatory mechanisms, and clinical application based on electrospinning technology in the future.