The invention provides a motherboard and an interface control method of a memory slot thereof. The motherboard includes a plurality of slot groups, a bus, and an interface controller. Each of the slot groups includes a first memory slot and a second memory slot connected with the bus. The first memory slot and the second memory slot form two different access addresses. The interface controller transmits a plurality of pin control signals to the corresponding slot groups to make the two access addresses of the first memory slot and the second memory slot of a using slot group of the slot groups different from the two access addresses of the first memory slot and the second memory slot of each of the other slot groups. Then, the interface controller accesses the using slot group via the bus.