Prolyl 4-hydroxylases (P4Hs) are important regulators of the extracellular matrix in cancer by promoting collagen deposition. These enzymes catalyze the hydroxylation of proline residues in collagen, facilitating the formation of its triple-helical structure and enhancing thermal stability. However, several collagen-independent mechanisms of P4Hs have recently been identified in tumor pathogenesis. The P4H family includes catalytic P4HA isoforms and the chaperone protein P4HB/protein disulfide isomerase, each exhibiting distinct tumor type-specific expression patterns. For instance, P4HA1 has been reported to promote glycolytic reprogramming, at least partly through indirect stabilization of hypoxia-inducible factor 1α. P4Hs can hydroxylate a variety of proteins, thereby modulating their functions. Additionally, P4H isoforms may also interact with post-translational regulatory networks, both as modifiers of selected substrates and as proteins that are subject to regulatory modifications themselves. Furthermore, these enzymes are involved in regulating cell death, including ferroptosis and cuproptosis, as well as modulating endoplasmic reticulum stress responses. The activity of P4Hs is tightly regulated by complex networks of upstream transcription factors and downstream signaling pathways. This review summarizes both collagen-dependent and collagen-independent mechanisms of P4H-related proteins and evaluates their potential associations with tumor prognosis and immunotherapy response based on current preclinical and translational evidence. Finally, the therapeutic potential of targeting P4H-related pathways is discussed, together with key limitations, including limited isoform selectivity, off-target effects, disruption of normal collagen homeostasis, and insufficient clinical validation. Emerging targeted-delivery and microenvironment-responsive strategies are also considered as potential approaches to improve spatial specificity and therapeutic safety.