A previous laboratory study using Haemaphysalis longicornis Neumann (Acari: Ixodidae) ticks of North American origin showed that larvae could acquire the Lyme disease spirochete, Borrelia burgdorferi sensu stricto (s.s.) (Spirochaetales: Spirochaetaceae) while feeding to completion on infected mice. However, the infection was lost during the molt to the nymphal stage. Nonetheless, questing H. longicornis nymphs and adults collected by drag sampling in the northeastern United States have been reported infected with B. burgdorferi s.s. DNA; occasionally these ticks appeared to be partially engorged. This raises the question of whether H. longicornis ticks can (i) acquire B. burgdorferi s.s. during an interrupted, partial blood meal on an infected host and (ii) transmit spirochetes while completing the blood meal on a second host. In this laboratory study, we demonstrated that H. longicornis nymphs could acquire B. burgdorferi s.s. from infected Mus musculus mice during a partial blood meal. Borrelia burgdorferi s.s. was detected by a multiplex polymerase chain reaction amplicon sequencing assay in 2 of 32 (6.3%) nymphs allowed to remain attached to infected mice for 48 h but, paradoxically, not in any of 25 nymphs that remained attached to infected mice for 72 h. Unfortunately, due to the low percentage of infected nymphs, we were not able to examine if such partially fed, infected nymphs were able to transmit B. burgdorferi s.s. while completing their blood meal on a second, naïve host.