Simple SummaryNutrition management strategies are closely related to body development and health, and feeding frequency affects pig feed intake, feed efficiency, body composition, and growth performance. However, the effect of feeding one time daily and two times daily on the intestine has been given less attention. In this study, we investigated the transcriptomic responses induced in the livers and jejunal mucosa of growing pigs by daily feeding schedules. We found that when compared with feeding once daily, two times feeding had no significant effect on the growth performance of growing pigs with the same average daily feed intake. A two meals regimen reduced the concentration of triglycerides in serum and liver, affected the body metabolism by promoting lipid transport, lipogenesis, fatty acid oxidation, chylomicron formation and transport, gluconeogenesis, and inhibiting adipocyte differentiation. These findings support the idea that different feeding regimens could affect lipid metabolism and can be effective in nutritional strategies against metabolic dysfunction.Feeding frequency in one day is thought to be associated with nutrient metabolism and the physical development of the body in both experimental animals and humans. The present study was conducted to investigate transcriptomic responses in the liver and jejunal mucosa of pigs to evaluate the effects of different feeding frequencies on the body’s metabolism. Twelve Duroc × Landrance × Yorkshire growing pigs with an average initial weight (IW) of 14.86 ± 0.20 kg were randomly assigned to two groups: feeding one time per day (M1) and feeding two times per day (M2); each group consisted of six replicates (pens), with one pig per pen. During the one-month experimental period, pigs in the M1 group were fed on an ad libitum basis at 8:00 am; and the M2 group was fed half of the standard feeding requirement at 8:00 am and adequate feed at 16:00 pm. The results showed that average daily feed intake, average daily gain, feed:gain, and the organ indices were not significantly different between the two groups (p > 0.05). The total cholesterol (T-CHO), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) concentrations in the serum, and the TG concentration in the liver in the M2 groups were significant lower than those in the M1 group, while the T-CHO concentration in the liver were significant higher in the M2 group (p < 0.05). Jejunal mucosa transcriptomic analysis showed the gene of Niemann-Pick C1-Like 1 (NPC1L1), Solute carrier family 27 member 4 (SLC27A4), Retinol binding protein 2 (RBP2), Lecithin retinol acyltransferase (LRAT), Apolipoprotein A (APOA 1, APOA 4, APOB, and APOC 3) were upregulated in the M2 group, indicating that fat digestion was enhanced in the small intestine, whereas Perilipin (PLIN1 and PLIN2) were downregulated, indicating that body fat was not deposited. Fatty acid binding proteins (FABPs) and Acetyl-CoA acyltransferase 1 (ACAA1) were upregulated in the M2 group, indicating that two times feeding daily could promote the oxidative decomposition of fatty acids. In conclusion, under the conditions in this study, the feeding frequency had no significant effect on the growth performance of pigs, but affected the body’s lipid metabolism, and the increase of feeding frequency promoted the fat digestion in the small intestine and the oxidative decomposition of fatty acids in the liver.