Landscape connectivity is fundamental to maintain the biodiversity in fragmented areas. Several metrics are used to evaluate landscape connectivity. However, metrics based on graph theory have been widely applied by integrating the connected habitat area that exists within the fragments (intrapatch connectivity) and connections between different fragments (interpatch connectivity) in a single index. Functional connectivity depends on permeability and heterogeneity of the matrix and, therefore, it can be modify by land use change. The expansion of agriculture has been an important impact factor on biodiversity. Sugarcane is the main crop in São Paulo state and its intense expansion can affect the connectivity due to the change on landscape configuration. The aim of this study was to evaluate the influence of sugarcane expansion on connectivity for medium and large mammals which require forest habitat in a portion of Novo Horizonte (SP), between 1995 and 2015. Circuit theory was used to estimate effective distance between fragments and to model connectivity in non-preferential areas for these animals. Graph theory was applied to quantify and assess the effect of land use change on landscape connectivity and also to prioritize areas for conservation using the probability of connectivity index. For the mammals groups evaluated, the resistance level of the matrix increase due to sugarcane expansion negatively impacting the species’ dispersal. However, the landscape connectivity before the expansion of this crop was already low.