Intracellular pressure is one of the most fundamental mechanical factors, influencing cellular homeostasis and functions. Gaining insight into the intracellular pressure is essential for understanding fundamental cellular processes and developing therapeutic strategies. In this review, we provide an overview of the intracellular pressure, focusing on three key aspects including its regulation, characterization, as well as biological implications. We summarize representative experimental and modeling methods, highlighting their principles, strengths, and weaknesses. Meanwhile, we discuss how the intracellular pressure is generated and how it shapes the biological processes, particularly exemplified by cell division and migration. We highlight the necessity and significance of developing non-invasive and precise techniques and methods to characterize in vivo intracellular pressure under different circumstances. More light should be shed on intranuclear pressure and nuclear response to the intracellular pressure dynamics. In addition, further efforts should also be directed toward exploring the intrinsic relationship between intracellular pressure and other cellular activities.