The demand for injectable dermal filler has undergone significant growth with the rapid development of the beauty industry. Poly(lactic acid) (PLA) as a benefit of excellent biocompatibility and long-term promotion of collagen regeneration has been favored as a commonly used filler. However, the effects of chirality and particle size of PLA on the efficacy of dermal filler have not been studied. In this study, we prepared three kinds of microspheres (MSs) consisting of poly(D-lactic acid) (PDLA MS), poly(L-lactic acid) (PLLA MS), or meso-PLA (PDLLA MS) at 5,10 and 20 μm to reveal the different biological functions as dermal filler. Following intradermal injection into guinea pig, it was found that PLLA MS induced the slightest inflammation, and the level of pro-inflammatory cytokine IL-1β induced by PLLA MS is only 0.3 or 0.7-fold of that induced by PDLA or PDLLA MS, respectively. More importantly, PLLA MS significantly stimulated the regeneration of collagen, which was 1.4 or 1.1 times higher than those stimulated by PDLA MS or PDLLA MS, respectively. The size of PLA MSs did not affect the levels of inflammation and collagen regeneration. The results confirmed the superiority of PLLA as a dermal filler.