Pork is one of the most widely produced and consumed meats in the world, and it is also an important source of animal protein. The continuous rise in feed prices has forced the pig industry to consider adding cost-effective alternative feed to pig diets. In this study, we aimed to explore the beneficial effects of tuna dark muscle as a nutritional supplement on the growth performance, serum lipids and antioxidant levels of Holland mini-piglets, as well as on the odor and volatile substances of pork and the gut microbiota. Two-month-old male mini-piglets (n = 24) were fed a control diet or supplemented with either 2% (LD) or 4% (HD) tuna dark muscle for 8 weeks. The use of tuna dark muscle at low and high dosages significantly increased the average daily weight gain, but it showed no significant effect on organ indices or blood lipids. In addition, dark muscle treatment significantly increased the antioxidant capacity, characterized by increased SOD and GSH-Px activities, and it decreased the content of MDA in serum. Moreover, tuna dark muscle feeding shifted the odor of rib muscle and tendon meat away from that of the control group, while similar odor patterns were observed in the longissimus dorsi muscle. Among these volatile substances, hexanal, nonanal, and heptanal increased in response to dietary tuna dark muscle and were regarded as indispensable contributors to the feeding. Furthermore, dietary tuna dark muscle modulated the gut microbiota of the piglets, increasing the abundance of beneficial bacteria such as butyric acid-producing bacteria, and reduced the abundance of harmful bacteria. The feeding strategy reported in this study not only reduces the production costs of pork but also utilizes tuna processing by-products in an environmentally friendly way.