To study the effect of unloading followed by reloading on hindlimb bone mineral content (BMC), we used botulinum toxin A (Botox). We studied the timing and degree of recovery upon restoration of muscle function. We also tested to see if reaction to Botox injection occurred as a function of the degree of expression of connexin43 (Cx43). Sixteen mice were divided by gender and genotype; wild-type equivalent (Gja1+/flox) and heterozygous (Gja1+/-) mice were injected with a single dose of 2U/100 g Botox i.m. in the quadriceps, hamstrings, and posterior calf muscle groups (day 0). Regional BMC was monitored for 12 weeks in the injected and contralateral control limb. Significant bone loss was observed in the injected limb by week 2 in the Gja1+/flox mice, and by week 3-4 in the Gja1+/- mice. By week 12, BMC in the Botox-treated limb of both male and female wild-type and heterozygous mice was still at least 14% less than that of the noninjected limb. Cortical thickness and BV/TV were lower in the Botox femur compared to the noninjected femur. The heterozygous mice tended to show a slower response to Botox injection as reflected in a slower loss of BMC. Our results show that the rapid and profound bone loss following temporary muscle paralysis is not fully recovered upon restoration of muscle function within 12 weeks. These results underscore the significance of normal muscle function in the maintenance of bone mass.