This study examines the influence of fatigue and defensive pressure on the kinematic parameters of the three-point jump shot in basketball. Fourteen male collegiate basketball players (age: 21 ± 3 years old, body height: 186.35 ± 7.02 cm, body mass: 82.20 ± 10.99) participated in the study. Each participant performed three-point jump shots under four conditions: without defense, with defense, without defense after a fatigue protocol, and with defense after a fatigue protocol. Kinematic data were collected using the Xsens MVN inertial suit system and the OptoJump Next system. The analysis focused on various parameters including jump height, center of mass, release height, shoulder angle, and segment velocities. The repeated-measures ANOVA was used to observe the differences between each shot condition (fatigue, defense). Results indicated significant changes in the kinematic parameters due to both fatigue and defensive pressure. Fatigue notably changed shooting performance, affecting jump height and release mechanics. The defensive pressure altered shooting technique, leading to quicker ball release and higher release points. These findings highlight the importance of incorporating fatigue and defensive scenarios in training, suggesting that coaches develop more targeted training plans to improve performance under conditions of fatigue and defensive pressure.