Deloria was the grandson of Tipi Sapa (Black Lodge), also known as Rev. Philip Joseph Deloria, an Episcopal priest and a leader of the Yankton band of the Nakota Nation. Vine, Jr. was born in Martin, South Dakota, near the Oglala Lakota Pine Ridge Indian Reservation. He was first educated at reservation schools.

Deloria's father, Vine Sr. (1901–1990), studied English and Christian theology, became an Episcopal archdeacon and missionary on the Standing Rock Indian Reservation, to which he transferred the family's tribal citizenship. Deloria Jr.'s aunt was the anthropologist Ella Deloria (1881–1971).

Deloria graduated from Iowa State University in 1958 with a degree in general science. Deloria then served in the Marines from 1954 through 1956.

Deloria Jr. originally sought to be a minister, like his father, and in 1963 earned a theology degree from the Lutheran School of Theology in Rock Island, Illinois. Deloria earned a law degree from the University of Colorado in 1970.

"Mr. Deloria ... steadfastly worked to demythologize how white Americans thought of American Indians," wrote Kirk Johnson.

In 1964 Deloria was elected executive director of the National Congress of American Indians. During his three-year term, the organization went from bankruptcy to solvency, and membership went to 19 to 156 tribes.

While teaching in Bellingham, Washington, Deloria advocated for the fishing rights of local tribes. He worked on the legal case that led to the Boldt Decision in 1974, United States vs. Washington, which validated Indian fishing rights.

In 1969, Deloria published his first of more than twenty books, entitled Custer Died for Your Sins: An Indian Manifesto. This book became one of Deloria's most famous works. In it, Deloria addressed Indian stereotypes and challenged white audiences to take a new look at the history of American western expansionism. The book was released around the time that the American Indian Movement was gaining momentum, and the book helped draw attention to the Native American struggle. The book focused on the Native American goal of sovereignty without political and social assimilation, and stood as a hallmark of Native American Self-Determination at the time. The American Anthropological Association sponsored a panel in response to Custer Died for Your Sins. The book could be described as a good-natured polemic, combining scholarship, advocacy, and humor.

In 1995, Deloria argued in his book Red Earth, White Lies, that the Bering land bridge never existed, and that the ancestors of the Native Americans did not migrate to the Americas over such a land bridge, as has been claimed by many archaeologists. Rather, he asserted that the Native Americans may have originated in the Americas, or reached them through transoceanic travel, as some of their creation stories suggested. His views on the age of certain geological formations, the length of time Native Americans have been in the Americas, their possible co-existence with dinosaurs, etc. were very influential in the development of American Indian Creationism.

Deloria wrote and edited many subsequent books and 200 articles, focusing on many issues as they related to Native Americans, such as education and religion.