A cutter holding assembly has a holder, a cutter, and a tightening positioning assembly. The holder has a first positioning recess. The cutter is connected to the holder and has a second positioning recess. The tightening positioning assembly has a positioning block, two tightening elements, and an adjusting element. The positioning block is mounted in the first positioning recess of the holder and the second positioning recess of the cutter. The tightening elements are mounted in two opposite sides of the positioning block. The adjusting element is mounted in the positioning block at a position between the two tightening elements. The adjusting element has a conical abutting surface for pushing the tightening elements to move opposite to each other to abut the opposite side surfaces of one of the first and the second positioning recesses while axially moving. The cutter can be firmly connected to the holder.