He hailed from Maoling 茂陵 in the commandery Fufeng 扶風 (modern Xingping 興平, Shaanxi) and participated in an upring that his father Ma Teng 馬騰 and Han Sui 韓遂 initiated in the province of Liangzhou 涼州 (modern Gansu) during the reign of Emperor Ling 漢靈帝 (r. 167-189). Ma Teng later again declared his subservience to the Later Han dynasty 後漢 (25-220 CE), travelled to the capital and was appointed Chamberlain for the Palace Garrison (weiwei 衛尉). Ma Chao at that time took over the post of Determined General (pian jiangjun 偏將軍) in which he was responsible for the appeasement of the native tribes of the Qiang 羌 and Hu 胡 (Xiongnu 匈奴) in the northwestern region.
In 211, the warlord Cao Cao 曹操 (155-220) attacked the western region of Guanlong 關隴. Ma Chao and Han Sui immediately decided to resist Cao Cao and made their armies ready to await the warlord at the Tongmen Pass 潼關. Cao Cao was intelligent enough to wait until Ma Chao and Han Sui were at odds again because they suspected each other of treason. Han Sui began skirmishes with Cao Cao but was soon defeated. Ma Chao thereupon withdrew to the west and seized the territory of Guanlong and Liangzhou, killed the regional inspector (cishi 刺史), usurped local power and adopted the titles of General Conquering the West (zhengxi jiangjun 征西將軍) and military commander of Liangzhou.
Yet his power did not last for long. The official Yang Fu 楊阜 killed Ma Chao's wife and forced him to flee to the region of Hanzhong 漢中 (mountainous region between modern Shaanxi and Sichuan), where Ma Chao planned to join the Daoist sectarian rebel Zhang Lu 張魯 (d. 216 CE). Yet the two could not come to terms, and so Ma Chao joined the warlord Liu Bei 劉備 (161-222), who controlled the Sichuan Basin. Ma took over the siege of the city of Chengdu 成都 (modern Chengdu, Sichuan), where Liu Bei attacked the local power holder Liu Zhang 劉璋 (d. 219). Ma brought down the city and was highly praised by Liu Bei's strategist Zhuge Liang 諸葛亮 (181-234).
In 217, Ma Chao took part in the conquest of the Hanzhong region. When Liu Bei adopted the title of King of Hanzhong. Ma Chao was made General to the Left (zuo jiangjun 左將軍), and in 221 General of Cavalry (piaoji jiangjun 驃騎將軍), and was formally granted the title of regional governor (mu 牧) of Liangzhou.