Calibration is a strategy to compensate for the systematic errors of a mobile robot hence, to increase the accuracy of the robot localization. So far, various methodologies have been proposed for the calibration of wheeled robots, but the majority have focused on non-holonomic mobile robots, e.g., differential type, and holonomic ones have been less studied from this point of view. This paper presents an innovative approach for the calibration of a holonomic robot by introducing “Effective Kinematic Parameters” (EKPs). To estimate the EKPs of a holonomic mobile robot, some tests are proposed, and the variables of the inverse Jacobian equations are modified by defining and minimizing an appropriate cost function. In the following, a virtual holonomic robot is modeled with some unknown intentional errors in its parameters, which cause some error in its path tracking. Then, the methodology is applied to estimate its EKPs, and the simulation is fulfilled again. The results show incredible improvement in path tracking. The methodology is applied to a 3-wheeled Omni-directional mobile robot, and some experiments are performed in a laboratory environment to experimentally evaluate the approach. The outcomes approved the algorithm and showed great enhancement in the calibration of the robot compared to the previous researches. The methodology can be employed to calibrate other robotic systems as well.