In this work the effects of two different cyclic heat treatments (traditional route and new-designed route, named as Route 1 and Route 2, respectively) on microstructure and mechanical properties of Ti-V microalloyed steel were investigated. The results showed that the mean grain size initially decreased on execution of 1-cycle (~13.9 μm) and then remained almost constant during the subsequent cycles for Route 1. By contrast, the mean grain size decreased continuously with cyclic numbers and reached ~4.6 μm at 7-cycle for Route 2. For Route 1, with cyclic numbers UTS decreased continuously and AKU kept fluctuating due to the accumulation effect of temper softening and continuous fluctuation of RD (relative differences of grain sizes), respectively. For Route 2, the continuing decline of UTS with cyclic numbers could be ascribed to the increase of mean complex carbide size, while the AKU underwent a process of increasing and then decreasing with cyclic numbers and reached the maximum (~183 J) at 3-cycle as a result of joint influence of mean grain size and proeutectoid ferrite proportion.