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Mechanical properties are essential for the biological activities of cells, and they have been shown to be affected by diseases. Therefore, accurate mechanical characterization is important for studying the cell lifecycle, cell-cell interactions, and disease diagnosis. While the cytoskeleton and actin cortex are typically the primary structural stiffness contributors in most live cells, oocytes possess an additional extracellular layer known as the vitelline membrane (VM), or envelope, which can significantly impact their overall mechanical properties. In this study, we utilized nanoindentation via an atomic force microscope to measure the Young's modulus of Xenopus laevis oocytes at different force setpoints and explored the influence of the VM by conducting measurements on oocytes with the membrane removed. The findings revealed that the removal of VM led to a significant decrease in the apparent Young's modulus of the oocytes, highlighting the pivotal role of the VM as the main structural component responsible for the oocyte's shape and stiffness. Furthermore, the mechanical behavior of VM was investigated through finite element (FE) simulations of the nanoindentation process. FE simulations with the VM Young's modulus in the range 20–60 MPa resulted in force-displacement curves that closely resemble experimental in terms of shape and maximum force for a given indentation depth.