Aim: To study the effect of gum mastic, a natural resin, on the proliferation of androgen‐independent prostate cancer PC‐3 cells, and further investigate the mechanisms involved in this regulatory system, taking nuclear factor κB (NF‐κB) signal as the target. Methods: 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and a flow cytometer were used to detect the effect of gum mastic on the proliferation of PC‐3 cells. Then, reporter gene assay, RT‐PCR, and Western blotting were carried out to study the effects of gum mastic on the NF‐κB protein level and the NF‐κB signal pathway. The expression of genes involved in the NF‐κB signal pathway, including cyclin D1, inhibitors of κBS (IκBα), and phosphorylated Akt (p‐AKT), were measured. In addition, transient transfection assays with the 5 × NF‐κB consensus sequence promoter was also used to test the effects of gum mastic. Results: Gum mastic inhibited PC‐3 cell growth and blocked the PC‐3 cell cycle in the G1 phase. Gum mastic also suppressed NF‐κB activity in the PC‐3 cells. The expression of cyclin D1, a crucial cell cycle regulator and an NF‐κB downstream target gene, was reduced as well. Moreover, gum mastic decreased the p‐AKT protein level and increased the IκBα protein level. Conclusion: Gum mastic inhibited the proliferation and blocked the cell cycle progression in PC‐3 cells by suppressing NF‐κB activity and the NF‐κB signal pathway.