Nicholas was born in the Smolensk prefecture to Dimitry Kasatkin, a Russian Orthodox deacon. His mother died when he was five years old. He grew up in the church hierarchy: in 1857 he entered the Theological Academy in Saint Petersburg. On July 7, 1860 (July 19 in the Gregorian calendar), he became a monk and chose the name of Nicholas. Nicholas was ordained a deacon on July 12 (July 24) in the same year, on the feast day of Saints Peter and Paul. He was ordained a priest the next day, on the feast day of the Holy Apostles, the commemoration day of the Academy's Chapel of the Holy Apostles.
While at the consulate chapel, he converted three Japanese. Later, he moved to Tokyo, and began an extensive missionary effort. He bought property on a height in Kanda Surugadai for his headquarters which later became the site of the see of the Archbishop of Japan. Under his leadership, more than 250 communities were formed, and churches were built.
Nicholas was consecrated bishop on March 30, 1880, as Bishop of Revel, auxiliary to the Archdiocese of Riga. While Nicholas never visited the city, the parish of Revel supported his Japanese mission financially. In the Eastern Orthodox Church tradition, bishops sign with their parish, but Nicholas made his habit to sign as "Episcop (Bishop) Nicholai", without mentioning Revel.。 He was elevated to the dignity of Archbishop of All Japan by the Russian Holy Synod on April 6, 1907.
Even Emperor Meiji was impressed with his character, especially his Christian and diplomatic efforts between the Russian Imperial Household and the Japanese government. When the Russian Tsar Nicholas II was the Tsarevich under Alexander III, the young Nicholas II visited Japan and was injured during the Ōtsu Incident by a Japanese policeman. Bishop Nicholas made a great effort to resolve this incident.
Nicholas's study of Japanese was fruitful, allowing him to translate all liturgy books and many parts of the Bible including the whole of the New Testament and Psalms, most of Genesis and the Book of Isaiah with help from a Japanese Christian and scholar Nakai Tsugumaro who ran a kanbun private school Kaitokudo in Osaka.His translations are still used in the liturgy of Japanese Orthodox Church. Being fond of church singing, Kasatkin made a significant contribution in introducing this art to the Japanese.
He wrote a diary in Russian for years, recording the pastoral life of early Orthodox Church of Japan as well as his thought and observation of Meiji era Japan. His diary was believed to have been burned and lost in Great Kanto Earthquake but rediscovered by Kennosuke Nakamura, a Russian literary researcher, and published in 2004 as Dnevniki Sviatogo Nikolaia Iaponskogo (5 vols. St. Petersburg: Giperion, 2004). Nakamura translated the whole diary into Japanese and published with his commentary in 2007. as 『宣教師ニコライの全日記』(教文館、全9巻）.
There is a church which commemorates him in Maebashi, Gunma, built in 1974. There is also an Eastern Orthodox Church dedicated to Saint Nikolai of Japan in Moscow.