<p>An investigation on the tensile properties and strengthening mechanism of a dense 316L stainless steel (316LSS) material fabricated by laser powder bed fusion (L-PBF) has been conducted with varying heat treatment conditions. Deformation mechanisms of as-built and heat-treated samples were elucidated through multiscale microscopy characterizations. The cellular structure characterized by high density dislocation provided a strong barrier to the dislocation propagation, enhancing the yield strength of L-PBF 316LSS. Additionally, the accumulation of free dislocations at grain boundaries triggered the initiation of deformation twins and synergistically interacted with the dislocation wall to establish a three-dimensional network pinning structure, thereby effectively improving the continuous work hardening capability of the as-built sample. In contrast, the absence of cellular structure in HT 1000 samples resulted in a 21.8% reduction in yield strength and a 12% increase in elongation, exhibiting typical strength-ductility trade-off. The absence of cellular structure facilitated the formation of more deformation twins and contributed to the manifestation of the dynamic Hall–Petch effect. It effectively extended the work hardening regime of L-PBF 316LSS, thereby delaying the necking and enhancing its plasticity. Importantly, the Hall–Petch constant has been modified by analyzing the dependence of resolved shear yield strength, which originated from the cellular structure, on the inverse square root of cell size (<i>d</i>^−1/2). The modified Hall–Petch relationship accurately assessed the contribution of cellular structure to the yield strength of L-PBF 316LSS. The underlying strengthening mechanism of cellular structure was comprehensively revealed, and valuable insights for further optimization and enhancement of the mechanical properties of L-PBF 316LSS were offered.</p>