Transportation infrastructure, such as railways, roads and power lines, contribute to national and regional economic, social and cultural growth and integration. Kenya, with support from the Chinese government, is currently constructing a standard gauge railway (SGR) to support the country’s Vision 2030 development agenda. Although the actual land area affected by the SGR covers only a small proportion along the SGR corridor, a significant proportion of the area supports a wide range of ecologically fragile and important ecosystems in the country, with potential wider impacts. This study used a qualitative content analysis approach to gain an understanding and perceptions of stakeholders on the potential ecological impacts of the interactions between the SGR and the traversed ecological systems in Kenya. Three dominant themes emerged: 1) ecosystem degradation; 2) ecosystem fragmentation; and 3) ecosystem destruction. Ecosystem degradation was the most commonly cited impact at while ecosystem destruction was of the least concern and largely restricted to the physical SGR construction whereas the degradation and fragmentation have a much wider footprint. The construction and operation of the SGR degraded, fragmented and destroyed key ecosystems in the country including water towers, protected areas, community conservancies and wildlife dispersal areas. Therefore, we recommend that project proponents develop sustainable and ecologically sensitive measures to mitigate the key ecosystem impacts.