The analysis of the dynamics of the regional transport and logistics system of the Prydniprovsky economic region revealed many barriers to its effective functioning, which were divided into 10 groups: political, institutional, innovative, investment and financial, information, environmental, tariff, infrastructure, customs, and logistics. The forecast estimation of the indicators characterizing the development of the regional transport and logistic system in the Prydniprovsky economic region by using economic and mathematical tools was carried out, and on this basis tendencies of its functioning were revealed. It should be noted that forecast estimates should be considered forecasts-warnings. That is, provided that the insignificant level of labor productivity is maintained, as well as its reduction, the low return on capital investments in the coming years can significantly reduce the volume of transport (rail and road) in the Prydniprovsky economic region. Based on the regional development strategies of Dnipropetrovsk, Zaporizhzhia and Kirovohrad regions generalized, their shortcomings in creating a transport and logistics cluster were identified. In order to transform the regional innovation ecosystem, a conceptual approach to forming the transport and logistics cluster as an element of innovation infrastructure based on reasonable specialization was substantiated. It was proved that this structure should take the form of a partnership of business structures, research institutions, higher education institutions, institutions of logistics and innovation infrastructure, and government agencies. The structural scheme of interaction between the transport and logistics cluster and the innovation ecosystem was proposed to achieve the strategic goal of "Innovative development of the region on the basis of smart specialization" while implementing the strategic documents for the period till 2027. It was established that the development and implementation of basic conceptual provisions for forming a transport and logistics cluster would create appropriate conditions for increasing the volume and quality of transport and logistics services, for intensifying innovative development and implementing a qualitatively new regional pattern of sustainable development of the Prydniprovsky economic region, which must meet modern requirements for managing economic systems.