Sterols are essential lipophilic molecules that play pivotal roles in maintaining the structural integrity, fluidity, and functionality of eukaryotic cellular membranes. In addition to their structural roles, sterols participate in signal transduction, cellular differentiation, and environmental response mechanisms. While the fundamental framework of sterol biosynthesis is conserved among eukaryotes, microalgae exhibit remarkable diversity in sterol composition and biosynthetic routes, reflecting their wide phylogenetic diversity and ecological adaptation. In Chlamydomonas reinhardtii, sterol biosynthesis relies solely on the plastid-localized methylerythritol 4-phosphate (MEP) pathway, as the mevalonate (MVA) pathway is absent in this organism. However, other algal lineages, such as Ochrophyta (diatoms) and Haptophytes, retain the MVA pathway, as in land plants, indicating evolutionary diversity in isoprenoid precursor biosynthesis across microalgae. These intermediates feed into a conserved series of reactions producing squalene, followed by cyclization into cycloartenol via cycloartenol synthase (CAS) in most algae, although a minority of species utilize lanosterol synthase (LAS), which also serves as a central branching point for diverse sterol end products. Sterols in microalgae exist in both free and conjugated forms, including sterol esters that accumulate in cytosolic lipid droplets. Free sterols modulate membrane order, permeability, and domain formation, while esterified sterols serve as a reservoir that buffers sterol homeostasis during growth, stress, or metabolic shifts. This chapter provides a comprehensive overview of sterol biosynthesis in microalgae, highlighting key enzymes, metabolic intermediates, and pathway variations across taxa. In addition, we explore regulatory mechanisms underlying sterol production and remodeling in response to environmental cues such as nutrient availability, light, and temperature. Through integrating molecular insights with physiological and ecological perspectives, this chapter aims to deepen our understanding of sterol metabolism in microalgae and its potential exploitation in sustainable biotechnology.