Various cues from the microenvironment in which cells live can regulate cellular functions. In addition to biochemical cues, increasing evidence has demonstrated that mechanical cues (namely, substrate/matrix stiffness in this review) presented by the cell microenvironment are also critically important in regulating cellular functions. However, most studies on stiffness-regulated cellular functions mainly focus on 2D conditions, which might not be able to recapitulate the 3D microenvironment encountered by the cells in vivo. In contrast to the observations in 2D microenvironments, studies have already shown that cells respond differently to mechanical cues under 3D microenvironments. In this review, the mechanisms of cellular mechanosensing and mechanotransduction are briefly presented, followed by the introduction of the most studied 2D/3D platforms. The effects of substrate/matrix stiffness on cellular functions, including cell migration, spreading, proliferation, phenotype, and differentiation, under different dimensionalities are summarized and discussed. Finally, the persisting questions and future outlook are also proposed.