Background and Purpose: Gait analysis and the effects of levodopa on the gait characteristics in Mild parkinsonian signs (MPS) are rarely published. The present research aimed to (1) analyze the gait characteristics in MPS; (2) explore the effects of levodopa on the gait performance of MPS. Methods: We enrolled 22 inpatients with MPS and 20 healthy control subjects (HC) from Nanjing Brain Hospital. The Unified Parkinson's Disease Rating Scale was used to evaluate motor symptoms. Acute levodopa challenge test was performed to explore the effects of levodopa on the gait performance of MPS. The instrumented stand and walk test was conducted for each participant and the JiBuEn gait analysis system was used to collect gait data. Results: For spatiotemporal parameters: Compared with HC, the state before taking levodopa/benserazide in MPS group (meds-off) demonstrated a decrease in stride length (SL) (p≤0.001), an increase in SL variability (p≤0.001), and swing phase time variability (p=0.016). Compared with meds-off, the state after 1 hour of taking levodopa/benserazide in MPS group (meds-on) exhibited an increase in SL (p≤0.001), a decrease in SL variability (p≤0.001). For kinematic parameters: Compared with HC, meds-off demonstrated a decrease in heel strike angle (p=0.008), range of motion (ROM) of knee joint (p=0.011) and ROM of hip joint (p=0.007). Compared with meds-off, meds-on exhibited an increase in HS (p≤0.001). Bradykinesia and rigidity scores were significantly correlated with gait parameters. Conclusion: Although the clinical symptoms of the MPS group are mild, their gait damage is obvious and they exhibited a decreased SL and joints movement, and a more variable gait pattern. Levodopa had little effect on the gait performance of those individuals.