<p>Artificial insemination is a technique to transfer instrumentally male’s sperm into the female’s reproductive system. Artificial insemination of honeybee has been widely used in genetic improvement, disease-resistant breeding, and the conservation of superior bee strains. Compared with honeybees, bumblebees have been produced in commercial enterprises for annual breeding, but artificial insemination of bumblebee has not yet been widely used. In order to solve the key technical bottlenecks in artificial insemination of bumblebee, we explored thoroughly the characteristics of reproductive systems of <i>Bombus lantschouensis</i> and <i>B. terrestris</i> queens. We demonstrated that the insertion depth of the needle’s tip and the volumes of semen-diluent mixture were the key factors affecting the success rate of bumblebee artificial insemination; therefore, we established the reasonable technical parameters and optimized operation flow, which have been successfully used in artificially inseminated queens of <i>B. terrestris</i>, <i>B. lantschouensis</i>, <i>B. ignitus</i>, <i>B. eximius</i>, <i>B. melanopygus</i>, <i>B. patagiatus</i>, <i>B. breviceps</i>, and <i>B. montivagus</i>. The survival rate of queens inseminated exceeded 95%, and 80% of the inseminated queens successfully founded colonies. Additionally, a rapid and direct method to evaluate the success of artificial insemination was proposed by observing the color changes of the queen’s spermatheca in vitro. Our breakthroughs and improvements in artificial insemination of bumblebees provide strong technical support for commercial rearing, breeding, and scientific research, as well as critical support for the conservation of endangered bumblebee species.</p>