Lung cancer is the leading cause of cancer-related deaths globally and is histologically defined as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), with the latter accounting for 80% of all lung cancers. The 5-year overall survival rate for lung cancer patients is low as it is often discovered at advanced stages when potential cure by surgical resection is no longer an option. To identify a biomarker and target for lung cancer, we performed analysis of multiple datasets of lung cancer gene expression data. Our analyses indicated that the collagen-modifying enzyme Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1) is overexpressed in NSCLC. Furthermore, our investigation found that overexpression of enzymes involved in this pathway predicts poor outcome for patients with lung adenocarcinoma. Our functional studies using knockdown strategies in lung cancer cell lines in vitro indicated that P4HA1 is critical for lung cancer growth, migration, and invasion. Additionally, diethyl pythiDC (PythiDC), a small molecule inhibitor, decreased the malignant phenotypes of lung cancer cells. Moreover, we found that miR-124 regulates and targets P4HA1 in lung cancer cells. Thus, our study suggests that collagen-modifying enzymes play an important role in lung cancer aggressiveness. Furthermore, our studies showed that P4HA1 is required for lung cancer cell growth and invasion, suggesting its potential as a valid therapeutic target in lung adenocarcinoma.