RPL27 is linked to the development of various diseases including malignant tumors. RPL27 may play an oncogenic function in hepatocellular carcinoma (HCC), but this is unknown. So, the aim of this study was to investigate how the human liver cancer cell lines SNU449 and HepG2 responded to RPL27 knockdown in terms of proliferation and apoptosis. SNU449 and HepG2 were cultured and infected with shCon and shRPL27 lentiviral particles to induce RPL27 knockdown, and then RPL27 expression was detected using qPCR and Western blot. Cell proliferation was measured using CCK8, cell cloning, cell scraping, and transwell migration and invasion, while apoptosis was measured using flow cytometry (FCM). The qPCR revealed that mRNA expression of RPL27 decreased after knocking down RPL27 in cells. The CCK8 and cell cloning assay confirmed that knocking down RPL27 significantly reduced cell viability. The cell scratch assay and transwell assays showed that the proliferation rate decreased after knocking down RPL27. A substantial increase in apoptotic cells was discovered by FCM. According to WB, RPL27 knockdown increased the expression of Bax and Caspase-3 while decreasing the expression of bcl-2. The findings showed that RPL27 knockdown inhibited cell proliferation in SNU449 and HepG2 via inducing apoptosis, proving that RPL27 is a novel gene linked with HCC and is crucial for both proliferation and apoptosis. These outcomes imply that RPL27 may be a potential target for liver cancer diagnosis and therapy.