The paleohydrological condition in the Rostov depression (Yaroslavl region) has been the subject of many years of discussions. The ideas about the Holocene fluctuations of the Lake Nero level differ among researchers. We have studied the structure of bottom sediments and bottom topography in the deepest northeastern part of the lake. A bathymetric survey was carried out. Drilling with the selection of undisturbed columns, GPR profiling, radiocarbon dating and a set of lithological analyzes were performed. Stratigraphic unconformities in the structure of bottom sediments indicate a drop in the lake level during the Lateglacial and the early Holocene. The level dropped to 87 m asl, which is 7 m lower than the current water level in the lake. The size of the lake at this stage was reduced several times. From 9 to 6.5 ka BP a transgressive stage was established: the average level of the lake could have risen to 91–94 m asl, which is close to its modern level. From 6.5 to 2.4 ka BP a decrease in the level by 1–3 m below the current one is revealed, followed by a gradual increase in the level. The current level was reached 300–500 years ago. The main factor in the fluctuations in the level of Lake Nero in the Holocene is the change in the height of the runoff threshold, caused by the transformation of the Ustye, Veksa, and Kotorosl river sistems. This transformation was associated both with regional changes in fluvial activity and with the processes of self-development of river channels.