This study is the first to report the enhancement of cell migration and proliferation induced by in vitro microsecond pulsed electric field (μsPEF) exposure of primary bovine annulus fibrosus (AF) fibroblast-like cells. AF primary cells isolated from fresh bovine intervertebral disks (IVDs) are exposed to 10 and 100 μsPEFs with different numbers of pulses and applied electric field strengths. The results indicate that 10 μs-duration pulses induce reversible electroporation, while 100 μs pulses induce irreversible electroporation of the cells. Additionally, μsPEF exposure increased AF cell proliferation up to 150% while increasing the average migration speed by 0.08 μm/min over 24 h. The findings suggest that the effects of PEF exposure on cells are multifactorial—depending on the duration, intensity, and number of pulses used in the stimulation. This highlights the importance of optimizing the μsPEF parameters for specific cell types and applications. For instance, if the goal is to induce cell death for cancer treatment, then high numbers of pulses can be used to maximize the lethal effects. On the other hand, if the goal is to enhance cell proliferation, a combination of the number of pulses and the applied electric field strength can be tuned to achieve the desired outcome. The information gleaned from this study can be applied in the future to in vitro cell culture expansion and tissue regeneration.