When the Second Boer War began in late 1899, New South Wales raised multiple contingents which included detachments from the regiment. The first contingent left Newcastle on 14 November 1899 for Cape Town, South Africa, arriving on 13 December. Two detachments of the regiment served under General John French's Cavalry Division during the Second Boer War in 1899, composed of 141 men and 157 horses. The first detachment fought in the Battle of Slingersfontein on 16 January 1900 resulting in heavy casualties, and the first Australian casualty of the war. The unit then participated in the Battle of Poplar Grove and took part in occupying the towns of Driefontein and Bloemfontein. The regiment also took part in both the advance to Pretoria and the Battle of Zand River, as well as the subsequent Surrender of Pretoria on 1 June. They also saw action in the Battle of Belfast in 1901, and later withdrew from South Africa by late March of the same year.