The conventional two-dimensional (2D) culture is available as an in vitro experimental model. However, the culture system reportedly does not recapitulate the in vivo cancer microenvironment. We recently developed a tissueoid cell culture system using Cellbed, which resembles the loose connective tissue in living organisms. The present study performed 2D and three-dimensional (3D) culture using prostate and bladder cancer cell lines and a comprehensive metabolome analysis. Compared to 3D, the 2D culture had significantly lower levels of most metabolites. The 3D culture system did not impair mitochondrial function in the cancer cells and produce energy through the mitochondria simultaneously with aerobic glycolysis. Conversely, ATP production, biomass (nucleotides, amino acids, lipids and NADPH) synthesis and redox balance maintenance were conducted in 3D culture. In contrast, in 2D culture, biomass production was delayed due to the suppression of metabolic activity. The 3D metabolome analysis using the tissueoid cell culture system capable of in vivo cancer cell culture yielded results consistent with previously reported cancer metabolism theories. This system is expected to be an essential experimental tool in a wide range of cancer research fields, especially in preclinical stages while transitioning from in vitro to in vivo.