DNA replication is essential for maintaining genome integrity during cell division. However, endogenous and exogenous factors frequently induce replication stress, leading to replication fork stalling and genome instability if unresolved. Recent studies have identified multiple mechanisms that coordinate replication fork stabilisation, protection and restart, although their regulation and roles in disease remain incompletely understood. This review summarises the current understanding of the mechanisms regulating stalled replication forks during replication stress. It first outlines the major endogenous and exogenous sources of replication stress before discussing the key cellular responses that maintain fork integrity, including Rad3-related (ATR)-checkpoint kinase 1 (CHK1) signalling, replication fork reversal and replication fork protection. Finally, it reviews the relationship between replication stress and cancer development, together with recent advances in therapeutic strategies targeting replication stress response pathways. Future studies should clarify the interactions among replication stress response pathways and develop more effective therapeutic approaches that selectively target replication stress in tumour cells.