Sperm‑associated antigen 9 (SPAG9) is a biomarker and potential therapeutic target for several cancers; however, its involvement in liver cancer progression is not clear. The aim of the present study was to determine whether SPAG9 regulates proliferation of liver cancer. Immunohistochemistry and cell immunofluorescence were used to confirm the expression and the localization of SPAG9 in human liver cancer tissues and the liver cancer‑derived HepG2 cells. A small interfering RNA (siRNA) designed to target SPAG9 was transiently transfected into HepG2 cells using Lipofectamine™ 2000, and proliferation, apoptosis and cell cycle progression were analyzed using CCK‑8 assay and flow cytometry; western blotting was used to detect the expression of SPAG9, JNK, p38, MKK3 and MKK6, and co‑immunoprecipitation was used to assess the interaction between SPAG9 and JNK. SPAG9 was overexpressed in 16 out of 20 (80%) patients with liver cancer. The protein was localized in both the cytoplasm and nucleus of liver cancer cells obtained from patients and in HepG2 cells. Depletion of SPAG9 inhibited the proliferation of HepG2 cells, promoted apoptosis and arrested the cell cycle at the S phase. Moreover, cells deficient in SPAG9 had decreased expression of JNK, p38 and MKK3 compared to HepG2 cells not treated with an siRNA targeting SPAG9. In the present study, SPAG9 was revealed to regulate cell proliferation, apoptosis and cell cycle progression in liver cancer cells through the SPAG9/MKK3/p38 axis. This axis is a novel therapeutic target for liver cancer.