Natural killer (NK) cell-based cancer immunotherapy requires the stable large-scale expansion of highly pure and potent NK cells. However, conventional culture systems have limited expansion efficiency, and the key molecules regulating NK cell expansion remain unclear. In this study, we focused on factors expressed by feeder cells to identify critical regulators of NK cell expansion. NK cell expansion was successfully induced through feeder cell pretreatment using heat shock and chemotherapeutic drugs, and correlation analysis between stress ligand expression and NK cell expansion efficiency identified calreticulin as a key factor. Based on this finding, calreticulin-overexpressing K562 feeder cells (K562-CRT) were developed. NK cells expanded using K562-CRT feeder cells maintained high purity over an extended period and achieved more than 10,000-fold expansion following a single stimulation. In addition, enhanced and sustained cytotoxic activity was confirmed. These findings suggest that a calreticulin-based feeder cell strategy may provide an important platform for next-generation NK cell immunotherapy and large-scale NK cell manufacturing.