Orthorhombic molybdenum trioxide (α-MoO3) nanofibers with controlled morphology and diameter were synthesized by adjusting electrospinning and calcination conditions. Experimental design was utilized to vary several factors including solution properties, electrospinning parameters and environmental conditions to analyze their effects toward nanofiber morphology (i.e., diameter and bead density). Polyvinylpyrrolidone (PVP) content, which effects viscosity, predominated control morphology of nanofibers where low PVP content (i.e., 3.0 wt.%) yielded heavily beaded nanofibers with smaller diameter. The morphology of nanofibers was also tuned by adjusting electrospinning parameters (i.e., applied voltage and feed rate), where smallest nanofibers with minimum bead density was achieved at applied voltage of 8 kV with feed rate of 0.5 mL/h. The as-electrospun ammonium molybdate/ polyvinylpyrrolidone nanofibers were calcined to α-MoO3 nanofibers in air. The ramping rate significantly altered the morphology of α-MoO3 nanofibers where rapid thermal annealing with the ramping rate of 15 °C/s yielded smoother nanofibers whereas slower ramping rate yielded platelet-like structures. BET analysis showed an increase in specific surface area with decreasing average diameter (i.e., 6.7 m2/g for 125 nm-86.4 m2/g for 35 nm).