Abstract: To improve the stiffness and energy absorption performance of negative Poisson’s ratio honeycomb structures, a multilayer nested re-entrant hexagonal honeycomb was designed and fabricated by 3D printing using polylactic acid (PLA) and short carbon fiber-reinforced polylactic acid composite (CF-PLA). Quasi-static compression experiments and finite element simulations were conducted to investigate the effects of nesting layer number, connection mode, spacing, loading rate, gradient configuration, and array expansion conditions on the deformation mode, mechanical response, and energy absorption behavior. The results showed that the multilayer nested design significantly enhanced the structural stiffness and energy absorption capacity. With increasing nesting layer number, the deformation mode gradually evolved from local buckling to cooperative progressive collapse of multiple cells, and local instability was effectively delayed. Structural topology was found to play a dominant role in energy absorption, whereas material effects showed a clear configuration dependence. PLA performed better under stronger geometric constraints or when stable progressive collapse was required, while the high specific strength advantage of CF-PLA became more evident in multilayer nested configurations, at larger spacing, or under higher-order cooperative deformation. These findings demonstrate that the multilayer nested design is an effective strategy for improving the stability and energy absorption efficiency of negative Poisson’s ratio honeycomb structures and provides guidance for the design of lightweight, high-efficiency energy-absorbing structures.