OBJECTIVES: Worldwide, rotavirus infections cause approximately 453,000 child deaths annually. Two licensed vaccines could be life- and cost-saving in low-income countries where the disease burden is highest. The aim of our study was to estimate the total cost of implementing the rotavirus vaccine in the national immunisation programme of a low-income country. Furthermore, the aim was to examine the relative contribution of different components to the total cost. METHODS: Following the World Health Organization guidelines, we estimated the resource use and costs associated with rotavirus vaccine implementation, using Malawi as a case. The cost analysis was undertaken from a governmental perspective. All costs were calculated for a 5-years period (2012-2016) and discounted at 5%. The value of key input parameters was varied in a sensitivity analysis. RESULTS: The total cost of rotavirus vaccine implementation in Malawi amounted to US$ 18.5 million over a 5-years period. This translated into US$ 5.8 per child in the birth cohort. With GAVI Alliance financial support, the total cost was reduced to US$ 1.4 per child in the birth cohort. Approximately 83% of the total cost was attributed to vaccine purchase, while 17% was attributed to system costs, with personnel, transportation and cold chain as the main cost components. CONCLUSION: The total cost of rotavirus vaccine implementation in Malawi is high compared with the governmental health budget of US$ 26 per capita per year. This highlights the need for new financing opportunities for low-income countries to facilitate vaccine implementation and ensure sustainable financing.