Humāyūn inherited the hope rather than the fact of empire, because the Afghans and Rajputs were merely restrained but not reconciled to Mughal supremacy by the Mughal victories at Panipat (1526), Khanua (1527), and the Ghaghara (1529). Bahādur Shah of Gujarat, encouraged by Afghan and Mughal émigrés, challenged the Mughals in Rajasthan, and, although Humāyūn occupied Gujarat (Gujarāt) in 1535, the danger there ended only with Bahādur's death in 1537. Meanwhile, an Afghan soldier of fortune, Shēr Shah of Sūr (Shēr Shāh of Sūr), had consolidated his power in Bihar and Bengal; he defeated Humāyūn at Chausa in 1539 and at Kannauj in 1540, expelling him from India.

Humāyūn became a homeless wanderer, seeking support first in Sindh, then in Marwar, and then in Sindh again; his famous son, Akbar, was born there in 1542. Reaching Iran in 1544, Humāyūn was granted military aid by Shah Ṭahmāsp and went on to conquer (in what is now Afghanistan) Kandahār (1545) and to seize Kabul three times from his own disloyal brother, Kāmrān, the final time being in 1550. Taking advantage of civil wars among the descendants of Shēr Shah, Humāyūn captured Lahore (now in Pakistan) in February 1555, and, after defeating Sikandar Sūr, the rebel Afghan governor of the Punjab, at Sirhind, he recovered Delhi and Agra that July. Humāyūn was fatally injured by falling down the staircase of his library. His tomb in Delhi, built several years after his death, is the first of the great Mughal architectural masterpieces; it was designated a UNESCO World Heritage site in 1993.