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We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1∶1 mixtures of primary human hepatocytes (hHeps) from partial hepatectomy specimens and human adipose-derived stem cells (hADSCs) in concave microwells. The hADSCs in spheroids could compensate for the low viability and improve the functional maintenance of hHeps. Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by co-cultured spheroids was 10% higher on day 7, whereas urea secretion was similar, compared with mono-cultured spheroids. A quantitative cytochrome P450 assay showed that the enzymatic activity of co-cultured spheroids cultured for 9 days was 28% higher than that of mono-cultured spheroids. These effects may be due to the transdifferentiation potential and paracrine healing effects of hADSCs on hHeps. These co-cultured spheroids may be useful for creating artificial three-dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes.