Excessive intake of a high-calorie diet has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Several studies have investigated the effect of NAFLD on liver regeneration, but the effects of simple steatosis have been found to be inconsistent. We aimed to assess whether the initial phase of diet-induced lipid accumulation, induced by a diet containing moderate levels of fat, impairs liver regeneration after partial hepatectomy (PHx) of mice. Male ddY mice are prone to obesity, even when fed a relatively low-fat diet (FD). A model of early simple steatosis was created by feeding a 10% FD for 6 weeks to male ddY mice. Liver regeneration rate, DNA synthesis in hepatocytes, and DNA damage were then assessed. FD-fed mice had a slightly higher body mass (44.5 ± 2.6 grams vs. 48.1 ± 3.6 grams, P < 0.05), but not liver mass (2.55 ± 0.37 grams vs. 2.69 ± 0.26 grams). Lipid droplets appeared in FD-fed mouse hepatocytes and Oil Red O staining was five times as intense as in control mice. In FD-fed mice, liver regeneration rate after two-thirds PHx was lower and impaired DNA replication was also observed. FD induced the expression of the cyclin-dependent kinase inhibitor p27KIP, but not p21CIP. Moreover, greater histone 2A.X phosphorylation was observed, indicating that FD caused DNA damage. Even short-term feeding of a moderately high FD to male ddY mice results in lipid accumulation in hepatocytes, DNA damage, and greater expression of p27KIP, implying lower DNA synthesis.