A potential role for the gelatinases in adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using the gelatinase inhibitor tolylsam ((R)‐3‐methyl‐2‐[4‐(3‐p‐tolyl‐[1,2,4]oxadiazol‐5‐yl)‐benzenesulphonylamino]‐butyric acid). Differentiation of murine 3T3‐F442A preadipocytes (12 days after reaching confluence) into mature adipocytes in vitro was promoted in the presence of tolylsam (10–100 μmol/L). De novo development of fat tissue in nude mice injected with preadipocytes and kept on a high‐fat diet was significantly impaired following treatment with tolylsam (100 mg/kg per day for 4 weeks). Adipose tissue development in matrix metalloproteinase (MMP)‐2 deficient mice, kept on a high‐fat diet, was significantly impaired following administration of tolylsam (100 mg/kg per day for 15 weeks). This was associated with markedly enhanced metabolic rate. Treatment of MMP‐2‐deficient mice with tolylsam (100 mg/kg per day, 15 weeks) was associated with the preservation of collagen and a reduction in blood vessel size in adipose tissues in vivo. Furthermore, plasma levels of triglycerides and free fatty acids were reduced by tolylsam treatment of MMP‐2‐deficient mice (100 mg/kg per day, 15 weeks), whereas nutrient adsorption in the intestine was not affected. The results of the present study indicate that tolylsam promotes preadipocyte differentiation in vitro, but impairs adipose tissue development in vivo.