Nanofibrous nerve guides have gained huge interest in supporting the peripheral nerve regeneration due to their abilities to simulate the topography, mechanical, biological and extracellular matrix morphology of native tissue. Gum tragacanth (GT) is a biocompatible mixture of polysaccharides that has been used in biomedical applications. During this study, we fabricated aligned and random nanofibers from poly(l-lactic acid) and gum tragacanth (PLLA/GT) in various ratios (100:0, 75:25, and 50:50) by electrospinning. Scanning electron microscope demonstrated smooth and uniform nanofibers with diameters in the range of 733±65nm and 226±73nm for align PLLA and random PLLA/GT 50:50 nanofibers, respectively. FTIR analysis, contact angle, in vitro biodegradation and tensile measurements were carried out to evaluate the chemical and mechanical properties of the different scaffolds. PLLA/GT 75:25 exhibited the most balanced properties compared to other scaffolds and was used for in vitro culture of nerve cells (PC12) to assess the potential of using these scaffolds as a substrate for nerve regeneration. The cells were found to attach and proliferate on aligned PLLA/GT 75:25 scaffolds, expressing bi-polar neurite extensions and the orientation of nerve cells was along the direction of the fiber alignment. Results of 8 days of in vitro culture of PC12 cells on aligned PLLA/GT 75:25 nanofibers, showed 20% increase in cell proliferation compared to PLLA/GT 75:25 random nanofibers. PLLA/GT 75:25 aligned nanofibers acted as a favorable cue to support neurite outgrowth and nerve cell elongation compared with PLLA nanofibers. Our results showed that aligned PLLA/GT 75:25 nanofibers are promising substrates for application as bioengineered grafts for nerve tissue regeneration.