Animal-vehicle collision on the roads is a major cause of mortality of a wide range of animal taxa both within and around protected areas. This study has been conducted in the National Highway 715 (new) covering a continuous stretch of 64 km that passes through Kaziranga National Park (KNP) of Assam (India). The area falls between the boundary of KNP on its north and North Karbi Anglong Wildlife sanctuary on the south. The survey concentrated on the mortality study of four groups of vertebrates viz., amphibians, reptiles, birds, and mammals resulting from collisions with vehicles from October 2016 through September, 2017. A total of 6036 individual roadkills were registered, belonging to 53 species, 23 other taxa and 30 families of vertebrates, with herpetofauna being the most affected group followed by birds and mammals. The study evaluated seasonal variation in the overall roadkill pattern with highest mortality in the monsoon season 38.27% (n = 2310) and with peak casualties starting with the onset of rainfall (February and March) and during monsoons (July and August). The amphibian mortality was also found to be highest during the monsoon with 43.28% (n = 1575) of kills, as compared to the other three groups. NH-715 (new), therefore serves as a challenging passage for the animals, forming a major barrier for the faunal component of the Kaziranga-Karbi Anglong landscape. This study thus tried to reflect the often overlooked issue of roads and highways in terms of direct mortality of animals due to traffic and thereby can be helpful in understanding the seriousness of the situation and identifying prospective measures to be taken for sustainable coexistence of both animals and human.