Trans fatty acids (TFAs) are risk factors for cardiovascular diseases and have been suggested to play roles in various metabolic diseases, including non-alcoholic steatohepatitis (NASH). The present study aimed to assess the influence of TFAs on the development of NASH lesions. Male Harlan Sprague Dawley rats were fed a choline-deficient, methionine-lowered, L-amino acid-defined (CDAA) diet with or without TFAs for two, four, and 13 weeks. The CDAA diet caused hepatic steatosis, macrophage infiltration, and fibrosis. Further, it increased serum activities of aspartate and alanine aminotransferase, and upregulated inflammation- and fibrosis-related genes in the liver. TFAs enhanced or tended to enhance the serum ALT activity but did not affect other outcomes. In the present study using the CDAA rat model, NASH lesions were clearly induced; however, the effect of TFAs was minimal. In conclusion, TFAs may be a risk factor for NASH by enhancing hepatocellular injury. With the composition and amount used in the present study, TFAs did not affect hepatic steatosis, chronic inflammation, or fibrosis in rats fed with the CDAA diet. To assess the risk of TFAs for NASH, more comprehensive studies are warranted, using other compositions and/or amounts of TFAs.