The power transformer is important equipment for energy conversion and transmission in the power grid and its failure will bring significant economic losses to the power system. The hot-spot temperature is a primary factor affecting the reliability and service life of power transformers. In order to calculate the temperature distribution of oil-immersed transformer windings accurately, it is required to solve a multi-physical problem, in which the magnetic field and the fluid-temperature field are coupled. In the simulation of multi-physical field of transformer, many studies usually omit the turn-to-turn insulation of windings to simplify the oil-immersed transformer model. The purpose of this paper is to investigate the effect of the turn-to-turn insulation of windings in oil-immersed transformers on the simulation results. First, a 2D axisymmetric transformer model was established in ANSYS to study the effect of low-voltage winding turn-to-turn insulation on the magnetic field simulation results. The differences of eddy current losses in the simulation were also calculated in terms of the magnetic field simulation results. It is found that the turn-to-turn insulation does not affect the magnetic field distribution in evidence from the simulation results. Besides, the values of the magnetic field and eddy current loss appear significant differences only at both ends of the low-voltage winding. Second, the fluid-temperature field of the transformer was solved in Fluent and the turn-to-turn insulation was the controlled objective in the solution process with consideration of the difference in winding losses. The simulation results show that the turn-to-turn insulation of the winding does not change the oil flow distribution in simulation. The simulation model with turn-to-turn insulation appears a gradient in the temperature distribution of the discs and a rise in the overall temperature distribution. Moreover, this paper analyzes the possible reasons for the differences in the simulation results of the magnetic and temperature fields caused by the turn-to-turn insulation. Some results and conclusions in this paper can be used in related studies and designs.