Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2–4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.