A series of novel pleuromutilin derivatives containing 4-aminothiophenol moieties have been designed and synthesized as promising antibacterial agents against Methicillin-resistant Staphylococcus aureus (MRSA). The in vitro antibacterial activity of these semisynthetic derivatives against 4 strains of S. aureus (MRSA ATCC 43300, S. aureus ATCC 29213, S. aureus 144 and S. aureus AD3) was evaluated by the broth dilution method. Most of the synthesized derivatives displayed prominent in vitro activity (MIC ≤ 0.5 µg/mL). 12 Compounds possessed superior antibacterial activity against MRSA compared with valnemulin and retapamulin (MIC = 0.0625 µg/mL). Compounds 12, 16a, 16c and 19 exhibited the most effective antibacterial effect against MRSA (MIC = 0.015 µg/mL). Furthermore, the time-kill curves showed compounds 12 and 19 had a certain inhibitory effect against MRSA in vitro. Compounds 12 and 19 possessed longer PAE time (2.74 h and 3.11 h, respectively) than tiamulin (PAE = 2.04 h) against MRSA after exposure at 4 × MIC concentration for 2 h. Compounds 12 and 19 also displayed superior in vivo antibacterial efficacy (-1.20 log10 CFU/mL and -1.21 log10 CFU/mL, respectively) than tiamulin (-0.75 log10 CFU/mL) in reducing MRSA load in the mice thigh infection model. In addition, compound 19 had barely inhibitory effect on RAW 264.7 and 16HBE cells at 8 µg/mL. In molecular docking study, upon docking into the 50S ribosomal subunit, the binding free energy (ΔGb) of compound 12 and 19 was calculated to be -9.02 kcal/mol and -9.89 kcal/mol, respectively.