Rabbi Shmuel Mohilever was born on 27 Nisan 5584 (1824) in Hlybokaye near Vilna, the son of Rabbi Yehuda Leib, a scholarly merchant from a rabbinic family. Married at fifteen, he went three years later to study at the Volozhin yeshiva, where at eighteen he received semicha from Rabbi Yitzchak of Volozhin. He first traded in flax and, when his business failed, turned to the rabbinate.

He served as rabbi of his hometown and then of several communities in Poland and Lithuania, the most important being Suwałki (1860-1868), Radom (1868-1883) and Białystok (1883-1898). Having means of his own, he also ran a farm, experience that served him in his work for the Land of Israel. In his articles he argued for uniting "these two daughters of God, faith and knowledge".

His involvement with the Yishuv began in 1874. He was one of the founders of the Chovevei Zion movement and one of the first rabbis to actively support Jewish settlement in the Land of Israel. He notably encouraged the founding of the agricultural colony of Ekron (Mazkeret Batya) and won the support of Baron Edmond de Rothschild. He is regarded as one of the fathers of religious Zionism, paving the way for the Mizrachi movement.

He passed away in Białystok on 19 Sivan 5658 (1898).