Born in Milton, Pennsylvania. Hepburn studied at Princeton University, after which he entered the medical school of the University of Pennsylvania, receiving his M. D. degree in 1836. He then earned a master's degree from Princeton University. In 1834, he joined the Presbyterian Church in the USA (North) in Milton. He married Clara M. Leete in 1840 and went to China as a medical missionary the next year. While in Singapore, he acquired Yohane fukuin no den (The Gospel According to St. John), translated by Karl Friedrich August Gutzlaff, and sent it to the Presbyterian Church mission headquarters. He met Samuel Robbins Brown at the Morrison Memorial School in Hong Kong. From 1843, he worked as a medical missionary on Amoy Island, but he and his wife returned to the United States in 1845 due to her poor health.
On 18 Oct 1859, the Hepburns came to Japan and lived at Jobutsu Temple in Kanagawa. Brown arrived in Japan the next November, and they lived together in the temple. Hepburn opened a free medical clinic at Soko Temple but was ordered to close it. He then worked full-time on Japanese language research, focusing on creating an English-Japanese dictionary. In May 1863, he opened a free medical clinic at #39 Yokohama Settlement, and his wife opened an English academy in November. He excelled in the treatment of eye problems, but he also engaged in some internal medicine and surgery. He became widely know when he performed surgery for gangrene on the noted actor Sawamura Tanosuke, and operation recorded even in Ukiyoe. A number of well-known persons studied at Mrs. Hepburn's academy, including Hayashi Tadasu, Takahashi Korekiyo, Masuda Takashi (who founded Mitsui Bussan), Miyake Hide (who became surgeon general), and Hattori Ayao.
Eight years after arriving in Japan, Hepburn's Japanese language research culminated in the 1867 compilation and publication of his Japanese and English Dictionary, which included an English and Japanese index. As he had come to Japan to do evangelistic work and Bible translation, early on he and Brown began working together on translating the New Testament. Beginning in Mar 1874, he met with the translating committee at the Brown residence and participated in almost all of the work done by this committee. In 1882, Hepburn was appointed chair of the Old Testament translating committee, working with Guido Herman Verbeck and Philip Kemball Fyson. Concentrating all their energies, they finished the translation in 1887. The $2,000 he received from the sale of the publishing rights to his third edition of the Japanese-English dictionary he donated to Meiji Gakuin, and the Hepburn Building was erected. From 1889 to 1891, he served as head of the school.
In 1892 Hepburn built the Yokohama Shiro Church building and published Seisho jiten (Bible Dictionary) with Yamamoto Hideteru. On 22 Oct of the same year, he and his wife returned to the United States and resided in East Orange, New Jersey. In 1905, he received an honorary doctor of law degree from Princeton University. In a strange coincidence, the Hepburn Building at Meiji Gakuin burned to the ground the day he died. His wife preceded him in death on 4 Mar 1906 at the age of 88.