Objective To establish the H1299 cell line with HNRNPK downregulated by DOX and study the effect of HNRNPK downregulation on cell proliferation and apoptosis. Methods H1299 cells were infected with a packaged lentivirus. After 72 h, positive cells were selected by puromycin and expanded to a stable transgenic cell line. The expression efficiency of HNRNPK was detected by qPCR and Western blot. The effects of HNRNPK downregulation on cell proliferation and apoptosis were analyzed. Results The H1299 cell line with HNRNPK downregulated by DOX was established successfully. Downregulation of HNRNPK inhibited the proliferation of H1299 cells but did not affect apoptosis. Conclusions Downregulation of HNRNPK inhibits the proliferation of H1299 cells.