The prohibition of cross-dressing is primarily derived from the Old Testament, specifically in the book of Deuteronomy. This biblical injunction is often discussed within the context of maintaining distinct gender roles as ordained by God. The relevant scripture is found in Deuteronomy 22:5, which states: "A woman shall not wear man's clothing, nor shall a man put on a woman's clothing, for whoever does these things is an abomination to the LORD your God" .

This verse is situated within a section of Deuteronomy that addresses various laws intended to guide the Israelites in their daily lives and maintain holiness among God's chosen people. The prohibition against cross-dressing is understood to reflect a broader divine mandate for order and distinction within creation, as seen in the Genesis account of creation where God creates male and female (Genesis 1:27).

From a conservative Christian perspective, this prohibition is often interpreted as a reinforcement of the natural order and the distinct roles assigned to men and women. The act of cross-dressing is seen as a violation of these divinely instituted roles and an attempt to blur the lines that God has established. This interpretation is consistent with the broader biblical theme of maintaining purity and holiness, as well as the importance of adhering to God's commandments.

The New Testament does not explicitly address cross-dressing, but it does emphasize the importance of modesty and propriety in dress. For instance, 1 Timothy 2:9-10 advises women to dress modestly and with decency, while 1 Corinthians 11:14-15 discusses the natural distinctions between men and women, particularly in the context of hair length, which some interpret as an extension of the principle of maintaining gender distinctions.

Throughout church history, the prohibition of cross-dressing has been upheld by many Christian theologians and leaders as part of the moral law that transcends the cultural and ceremonial laws of the Old Testament. This view holds that the underlying principle of maintaining gender distinctions is rooted in the created order and is therefore applicable to all people at all times.

In contemporary discussions, the prohibition of cross-dressing is often debated in light of modern understandings of gender identity and expression. However, those adhering to a conservative interpretation of scripture maintain that the biblical mandate remains relevant and that the distinctions between male and female are part of God's intentional design for humanity.