Serum alanine aminotransferase (ALT) concentration is the most commonly used marker for hepatocellular injury. We investigated the suitable cutoff value of serum ALT for the diagnosis or prediction of fatty liver. In 1578 Japanese adults (1208 men, 370 women; 35–69 years of age) who visited our center both in 2000 and between April 2007 and March 2008 (2007–2008), serum ALT concentration was an independent predictor of fatty liver in men in 2000 and in both sexes in 2007–2008. A significant increase in the frequency of fatty liver was detected in participants with elevated serum ALT concentrations, and serum levels of ALT in 2000 were associated with fatty liver in 2007–2008 when the cutoff value was set at 30 IU/L in men and 19 IU/L in women. The frequency of fatty liver in 2007–2008 was significantly lower in participants without fatty liver in 2000 whose serum ALT decreased between 2000 and 2007–2008. Our results suggest that serum ALT might be not only an indicator of fatty liver but also a predictor of the regression of fatty liver, and cutoff values of serum ALT of 30 IU/L in men and 19 IU/L in women are suitable for the screening of fatty liver.