Background <p>Pyrrole-5-carboxylate reductase 1 (PYCR1) is a key enzyme involved in proline synthesis, which is closely related to the occurrence and development of liver cancer. In this study, we aimed to investigate the relationship and mechanism of PYCR1 on proline metabolism in hepatocellular carcinoma cells under hypoxic conditions.</p> Methods <p>GO and KEGG enrichment analyses were used to predict the biological function and possible mechanism of PYCR1 in hypoxic microenvironments. The energy metabolism kit and Western blot were used to detect the metabolic changes of SUN449 and Hep3B liver cancer cells under hypoxic conditions. The proliferative capacity of cells was evaluated using EDU incorporation assay and the Ki67 staining protocol. The apoptotic rates and Western blot were measured using flow cytometry. Additionally, Western blot analysis was used to examine the levels of proteins associated with relevant signaling pathways and pathway inhibitors.</p> Results <p>GO enrichment analysis showed that hypoxic PYCR1 might be related to amino acid metabolism. The 1% hypoxia model promoted proline synthesis and lactate dehydrogenase synthesis in hepatocellular carcinoma cells. Knockdown of PYCR1 reverses hypoxia-induced proline synthesis and NAD⁺/NADH ratio. PYCR1 promoted the proliferation of hepatocellular carcinoma cells under hypoxic conditions. PYCR1 knockdown reduces proliferation and increases apoptosis. Hypoxia can activate the MAPK/ERK/STAT3 pathway; knockdown of PYCR1 can inhibit the levels of ERK and STAT3 phosphorylated proteins, inhibit the proliferation of hepatocellular carcinoma cells, and the ERK inhibitor U0126 inhibits the expression of P-STAT3 in the downstream.</p> Conclusion <p>In summary, we report that hypoxia-mediated PYCR1 promotes proline synthesis in HCC, cell proliferation, inhibits apoptosis, and ultimately promotes tumor progression through the MAPK/ERK/STAT3 signaling pathway, suggesting that PYCR1 is a potential therapeutic target for HCC.</p>