Cholesterol accumulation plays a significant role in the pathogenesis of metabolic-dysfunction-associated steatotic liver disease (MASLD), yet changes in cholesterol homeostasis in MASLD remain insufficiently investigated. This study aimed to examine alterations in cholesterol synthesis and absorption by measuring plasma levels of endogenous cholesterol precursors (as markers of synthesis) and phytosterols (as indicators of absorption). A total of 124 MASLD patients and 43 healthy individuals were included. Our results showed higher plasma concentrations of lathosterol in the MASLD group ( = 0.006), in parallel with comparable concentrations of desmosterol ( = 0.472) and all analyzed phytosterols in both groups. Correlation analysis showed that both lathosterol and desmosterol were positively associated with non-invasive hepatic steatosis indices: FLI, HSI, and TyG index ( < 0.01, < 0.01, and < 0.05, respectively). Multivariate linear regression further confirmed that these synthesis markers remained significant predictors of FLI ( = 0.010), HSI ( = 0.013), and TyG index ( = 0.002), even after adjusting for other relevant variables. These findings indicate that MASLD is associated with a shift in cholesterol homeostasis towards enhanced endogenous cholesterol synthesis.