這兩個詞在用作關係代詞以引導定語從句時，在句中的功能和意義完全相同，有時可互換使用。例如：
The delegates that (或who) were prent at the ferenumbered fifty-five．
出席會議的代表共有55名。
但在大多數的場合下，that和who的用法有所不同，大致有下列幾點區別：
（一)在較古老的英語裏，that既可以用以指物，又可用以指人。它既可引導限製性定語從句，又可引導非限製性定語從句。在現代英語中，that大多數用以指物；who則專用以指人。that所引導的定語從句大都是限製性的，而who引導的從句可能是限製性的，也可能不是限製性的。例如：
This is the cock that crew in the morn，
Unto the farmer sowing his ，
That met the priest with his pen and ink-horn，
That married the man so tattered and torn，….
He who does not ri early never does a good day''s work．
Mr.Li, who has never done such a thing before, shall be pardoned.
（二)在用It is…，only和最高級形容詞修飾的名詞之後，不論是物還是人，用that比用who普遍得多。如：
This is not the only thing that she wants．
She is the best woman writer that has ever visited a．
（三)that不能像whom那樣直接放在介詞之後，必須把介詞移至句末。如：
That is the man that we were speaking about.
試比較：That is the man about whom we were speaking.
請注意，下麵兩個句子的含義並不相同：
The members of Hong Kong Translation Society that（或who) were prent numbered fifty-five.出席的香港翻譯學會會員共有55名。（言外之意，還有些會員沒有出席。) The members,who (不能用that)were prent，numbered fifty-five.出席的會員達55名。（意指該會的全部會員都已出席。）