Background: Lycium barbarum polysaccharide (LBP) is a water-soluble polysaccharide extracted from Lycium barbarum. LBP exhibits potential pharmacological activity, including anti-cancer activities. However, so far, the effect of LBP in combination with cisplatin (DDP) on the proliferation of human alveolar adenocarcinoma cell line (A549) of non-small cell lung cancer (NSCLC) has not been reported. Objectives: In this study, we aimed to investigate the effect of LBP combined with DDP on the proliferation of A549 cells. Materials and Methods: The cells were divided into four groups: control group, DDP group, LBP group, and LBP and DDP combined group and Three parallel experiments were set up in each group. The survival rate of A549 cells and the effects of DDP and LBP alone or in combination was detected using the cell counting kit-8 (CCK-8) method. The activity of superoxide dismutase (SOD) was determined by xanthine oxidase method. The content of glutathione (GSH) was determined by colorimetry. The content of malondialdehyde (MDA) was determined by thiobarbituric acid method. The apoptosis, cell cycle phase, and the level of reaction oxygen species (ROS) formed were detected by flow cytometry. The expression of apoptosis-related proteins such as Bcl-2, Bax, and caspase-3 and cell cycle–related proteins namely, CDK4, cyclin D1, and p-Rb were detected via Western blot analysis. Results: DDP (≥ 6 mg/L) and LBP (≥ 8 mg/L) alone significantly inhibited the proliferation of A549 cells (P < 0.01), and LBP combined with DDP significantly enhanced the inhibitory effect on the proliferation of A549 cells (P < 0.01). DDP significantly reduced the activity of SOD and the level of GSH (P < 0.01), and significantly increased the level of MDA and ROS (P < 0.01). Compared with DDP group, the activity of SOD and the content of GSH and MDA in LBP and DDP combined group did not change significantly (P > 0.05), but the content of ROS decreased significantly (P < 0.01). DDP and LBP, alone and in combination, significantly promoted the cellular apoptosis (P < 0.01). They also significantly downregulated the expression of Bcl-2 and upregulated the expression of Bax and caspase-3, and the level of Bax/Bcl-2 was significantly increased (P < 0.01). DDP blocked the A549 cells in S phase, whereas LBP blocked the cells in G2/M phase. DDP and LBP combination significantly downregulated the expression of cell cycle regulatory proteins namely, CDK4, cyclin D1, and p-Rb (P < 0.05). Conclusion: LBP in combination with DDP inhibited the proliferation of A549 cells, and this combined effect is related to the expression of apoptosis-related proteins and the regulation of cell cycle mediated by cyclin D1-CDK4-Rb pathway.