Land use/cover changes impact species distribution via changing biological habitats. Land use change could reduce the number of larger habitat patches, which makes species survival highly depending on the connectivity of habitat patches. However, in previous studies on land use change and its impacts on ecosystem, species selection was based on individual species, focus species, and "virtual species" without exact species name by setting different distance thresholds to represent species with different dispersal capacity. Few studies have been conducted on the impacts of land use type's changes on real-world multi-species. Therefore, the objective of this study was to assess the impacts of overall and land use changes on real-world multispecies' habitat changes, particularly changes in landscape connectivity. We attempted to answer the following research questions:(1) how to measure the positive or negative effects of overall and land use changes on landscape connectivity of multispecies with different ecological requirements; (2) how to spatialize the impacts to identify priority of the protected areas? Gongyi City of Henan Province, a typical agricultural area in the middle reaches of the Yellow River, was selected as a case study area. The mammals lived in the region, Siberian Weasel (Mustela sibirica), Tolai Hare (Lepus tolai) and Yellow-throated Marten (Martes flavigula), were selected as the target species. Each species dispersal capacity was determined as 3 km, 7 km, and 7 km respectively according to their biological and ecological needs. Based on geographic information system technology, the land use changes in the study area from 1990 to 2011 were analyzed and were divided into 121 land use change types. Two connectivity indicators, the probability index of connectivity (PC) and the importance value (percentage of the variation in PC, dPC_k), were calculated. Then, the spatial distribution map of connectivity degree was constructed by using the inverse distance weight function. The results showed that great changes have taken place among 121 land use types from 1990 to 2011. The overall land use changes resulted in the increase of landscape connectivity (representing by the PC), with an increase of 22.22%, 25.64% and 45.46% to Siberian Weasel, Tolai Hare and Yellow-throated Marten, respectively. There was a difference between the PC calculated from overall and land use types changes. The impact of each land use type change on the PC was different. The increase and decrease of the PC were closely related to the increase and decrease of habitat patches area, respectively. From the spatial distribution map of multi-species landscape connectivity, the key areas of species conservation could be well identified. The study results highlighted the importance of habitat area and habitat connectivity for the biological conservation. This methodology used in this study was useful for conservation planning and landscape management, especially to spatialize the impacts of land use change on multi-species landscape connectivity.