Protected Areas networks should protect the native ecosystems from the negative influence of alien species introduction. We select a Protected Area (Mordovia State Nature Reserve, IUCN category: I) in European Russia (Republic of Mordovia) as a focal point of this study. Alien floras have never been compared between the Mordovian regional districts (managed areas) and Protected Areas. We aimed to compare the native vs. alien plant species proportion between the flora of the Mordovia State Nature Reserve and 23 floras of the regional districts based on the actual floristic check-lists of both Mordovia State Nature Reserve (at present, more than 800 species) and the whole Mordovia (at present, more than 1470 species), as well as the set of the published additions to the flora of the region and Protected Area. The temporal comparison will be made also between data of 2010 and 2021. We hypothesized that, contrary to the widely known fact on the intactness of Protected Areas, the Mordovia State Nature Reserve is characterized by a high proportion of alien plant species due to various anthropogenic influences. The obtained results demonstrated that in the Mordovia State Nature Reserve, the alien species proportion before starting intense botanical studies was lower (10.9%) than in floras of certain managed areas (17.2% in the Kochkurovo district, 17.0% in the Bolshie Berezniki district, or 22.4% in the Ardatov district). At the same time, additional studies have resulted in a considerable increase in the alien species proportion in the flora of the Protected Area (19.8%) in comparison with data of the 2010 year. This indicates both the initial underestimation of the Protected Area's flora and the importance of additional purposeful investigations to obtain complete data on the flora composition. A comparison of our results with the updated data for the Mordovian districts' floras is presented and discussed in the context of the current knowledge of the flora of the Mordovia State Nature Reserve and the Republic of Mordovia as a whole.