Background Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing various biological research. Methods and results The LRM cell culture developed from the muscle tissue of L. rohita was used to study the in vitro applications. The developed muscle cells were maintained in a Leibovitz‘s-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF at 28 o C temperature. The LRM cells showed fibroblastic-like morphology and was authenticated by sequencing mitochondrial gene 16S rRNA. The expression of myogenic regulatory factors (MRFs) was studied in different stages of LRM cells; however, the expression patterns varied at different passages. The MEF2A, Mrf-4, and Myogenin expressions were higher in passage 25, while the expression of MyoD was maximum in passage 15, and the expression of Myf-5 was highest in passage 1. The transfection efficiency of LRM cells revealed 14 % of the GFP expression with a pmaxGFP vector DNA. The LRM cells were susceptible to the extracellular products prepared from Aeromonas hydrophilla and Edwardsiella tarda . The acute cytotoxicity of six heavy metals (Hg, Cd, Zn, Cu, Pb, Ni) was assessed in LRM cells by a dose-dependent manner in comparison to IC 50 values obtained from MTT and NR assays. A revival rate of 70–75% was achieved when the LRM cells were cryopreserved at − 196 °C using liquid nitrogen. Conclusion The developed muscle cells serve as an functional in vitro tool for toxicological and biotechnological studies.