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To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the <i>Ppar</i>γ, <i>Ap2</i> and <i>Leptin</i> genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for <i>Ppar</i>γ expression; and (4) a significant downregulation in <i>Col1</i> and <i>Fn</i>, and a significant upregulation in <i>Col4</i>, <i>Col6</i>, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of <i>Ppar</i>γ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of <i>Ap2</i> were downregulated (2D, 3D) and <i>Leptin</i> was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of <i>Col1</i>, <i>Col4</i> was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.