The invention relates to a tightening and adjusting assembly for a steering system of a bicycle. The steering system generally includes a head tube, a steering tube rotatably mounted within the head tube by means of upper and lower headsets, and a handlebar stem tube mounted around the steering tube. The tightening and adjusting assembly has a preload member, an anchoring member and a tightening and adjusting set. The preload member is engaged with a top end of the handlebar stem tube. The anchoring member is abutted against a bottom end of the steering tube. The tightening and adjusting set is disposed in the interior of the steering tube and between the preload member and the anchoring member to draw the preload and anchoring members together and thus preload the upper and lower headsets of the steering system.