In this work, the paraffin oil was first molecularly distilled into two mineral oil saturated hydrocarbons (MOSH) subfractions with their main carbon number range of C 20−30 (84.58%) and C 30−50 (89.65%), which were determined using a liquid chromatography−gas chromatography−flame ionization detector (LC-GC-FID) technique. Then, both female and male Fischer 344 (F344) rats were fed with the diets containing one of these two MOSH subfractions at the low (1.5 g/kg) or high dose (15 g/kg) for 6 months. It was found that the exposure of MOSH C 20−30 subfraction could result in a significant increase of spleen weight index (4.64 ± 0.18 mg/g for female and 3.79 ± 0.31 mg/g for male rats) (P < 0.05), and irregular shape of white medulla, and the macrophage infiltration in mesenteric lymph node for both female and male rats, especially for the 15 g/kg MOSH C 20−30 -fed rats. Meanwhile, both MOSH C 20−30 and C 30−50 subfraction promoted inflammation and oxidative stress and had a negative effect on the immune function in both female and male rats. The metabolomics results further supported these observations, and indicated the down-regulation of cysteine and methionine metabolism, and glutathione metabolism pathway in female and male rats, respectively.