(1) Background: The purpose of this study was to investigate the effects of low-dose non-thermal atmospheric plasma (NTP) on the proliferation and migration of human immortalized keratinocytes (HaCaT cells) and its molecular mechanisms. (2) Methods: The effects of NTP on HaCaT cells were detected by cell viability, wound healing assay, cell cycle, mitochondrial membrane potential detection, and western blot, and the role of reactive oxygen species (ROS) content in low-dose NTP was explored. (3) Results: Results showed that the proportion of cells in G1-S phase transition, cell migration ability, and the expression of Cyclin D1 and STAT3 reached the peak at 10 s treatment group, while the cell viability and the expression of PI3K, AKT, mTOR, ERK, WNT, and β-catenin proteins reached the peak at 15 s treatment group. (4) Conclusions: These results manifested that ROS produced by low-dose NTP promoted the proliferation of HaCaT cells by activating the PI3K/AKT/mTOR signaling pathway and also promoted the migration by activating the WNT/β-catenin signaling pathway. Therefore, these results will be useful for the application of low-dose NTP in the treatment of wound healing.