During Mary's absence, Scotland underwent a Protestant Reformation led by figures like John Knox. This created a religiously divided nation that Mary would return to as a Catholic Queen.

Mary returned to a Scotland that had officially embraced Protestantism. She was allowed to practice her Catholic faith privately, but the tension between her religion and the dominant Protestantism was evident.

Mary initially practiced religious tolerance, hoping to maintain peace and stability. However, this policy was criticized by hardline Protestants like Knox, who saw Catholicism as idolatry and a threat to the true faith. Religious riots and conflicts occurred during this period.

Mary's opponents used her Catholicism as one justification for her deposition. Her marriage to Bothwell, suspected of murdering Darnley, further fueled religious fervor against her.

In England, Mary became a symbol for Catholic resistance against Elizabeth I. Catholic powers, like Spain, saw her as a potential replacement for Elizabeth, leading to plots and conspiracies.

The Papal Bull excommunicating Elizabeth I further encouraged Catholic plots against her and solidified Mary's role as a potential Catholic replacement.

The Babington Plot was motivated, in part, by the desire to restore Catholicism to England. Mary's perceived involvement in the plot, even indirectly, sealed her fate.