Commissioned in the British Indian Army in 1844, he served in the Punjab and travelled in the Himalayas and Tibet. In April 1855, as a member of Richard Burton's (Burton, Sir Richard) party attempting to explore Somaliland, Speke was severely wounded in an attack by the Somalis that broke up the expedition. In December 1856 he rejoined Burton on the island of Zanzibar. Their intention was to find a great lake said to lie in the heart of Africa and to be the origin of the Nile. After exploring the East African coast for six months to find the best route inland, the two men became the first Europeans to reach Lake Tanganyika (February 1858). During the return trip, Speke left Burton and struck out northward alone. On July 30 he reached the great lake, which he named in honour of Queen Victoria.

Speke's conclusion about the lake as a Nile source was rejected by Burton and was disputed by many in England, but the Royal Geographical Society, which had sponsored the expedition, honoured Speke for his exploits. On a second expedition (1860), he and James Grant mapped a portion of Lake Victoria. On July 28, 1862, Speke, not accompanied by Grant for this portion of the journey, found the Nile's exit from the lake and named it Ripon Falls. The party then tried to follow the river's course, but an outbreak of tribal warfare required them to change their route. In February 1863 they reached Gondokoro in the southern Sudan, where they met the Nile explorers Samuel Baker and Florence von Sass (who later became Baker's wife). Speke and Grant told them of another lake said to lie west of Lake Victoria. This information helped the Baker party to locate another Nile source, Lake Albert.

Speke's claim to have found the Nile source was again challenged in England, and, on the day he was to debate the subject publicly with Richard Burton, he was killed by his own gun while hunting. Accounts of his explorations were published in 1863 and 1864.