To investigate the effect of Serine-Arginine protein kinase 1 (SRPK1) on tumor stemness in HepG2 cells. Methods GEPIA2 (gene expression profiling interactive analysis 2) database was used to obtain the data about LIHC (liver hepatocellular carcinoma) in TCGA (The Cancer Genome Atlas) and analyze the difference in SRPK1 expression between liver cancer and normal samples, as well as the relationship with patient’s survival time and clinical stage. TIMER (Tumor Immune Estimation Resource) database was used to explore the correlation between SRPK1 expression and infiltrated immune cells in liver cancer tissues. HepG2 cells with overexpression and inhibition of SRPK1 protein were constructed and divided into the SRPK1 group, Vector group, shRNA group, and Scramble group. The protein level of SRPK1 was detected by Western blot. Sphere formation assay was used to detect the spheroidization ability of tumor cells, and flow cytometry was used to detect the proportion of side population (SP) cells in the cell population. The mRNA levels of tumor stemness markers, including Nanog, Oct4, CD133, and Bmi1, were compared by real time PCR. GSEA was used to evaluate the relationship between SRPK1 and the Wnt/β-catenin pathway in LIHC. Results TCGA data showed that the expression of SRPK1 mRNA in LIHC tissues was significantly higher than that of normal controls (P＜0.05), and there was a difference in the expression of SRPK1 mRNA in various stages of patients (P＜0.05). The overall survival of patients with high expression of SRPK1 was significantly poorer than those with low SRPK1 expression (P＜0.05). The expression of SRPK1 was significantly correlated with the infiltration levels of B cells, CD8+T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in liver cancer tissues (P＜0.05). Western blot showed that the SRPK1 protein in the SRPK1 group was higher than that in the Vector group (P＜0.01). SRPK1 group was higher than the Vector group in Tumor cell sphere formation efficiency (SFE), SP cell ratio, and mRNA of Nanog, Oct4, CD133, and Bmi1 (P＜0.05). Oppositely, the shRNA group indexes were lower than those of the Scramble group. GSEA indicated that high SRPK1 expression was positively correlated with Wnt/β-catenin pathway activation in LIHC (P＜0.05). Conclusion SRPK1 can promote tumor stemness acquisition in HepG2 cells.