The anti-proliferative effects of 5-methylquinolinium (5MQ) of nicotinamide N-methyltransferase (NNMT) have not been previously investigated on a cervical cancer cell line. NNMT is a metabolic enzyme that is correlated with tumour progression and metastasis. 5MQ is a small molecule inhibitor of NNMT. 0.1–500 μM of 5MQ was tested on the HeLa epithelial cervical cancer cell line. Cell viability was assessed with the MTT test. TWIST, ZEB1, SERPIN1, SIRT1, CD16, mRNA and various protein expression levels were analysed with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively. 5MQ significantly inhibited HeLa cell proliferation in a concentration and time-dependent manner. Increased cell shrinkage, loss of cellular adhesions and apoptotic bodies were observed in HeLa cells after 5MQ treatment. Following treatment with 5MQ, ZEB1, SIRT1, CD16 mRNA levels were increased while TWIST and SERPIN1 mRNA levels were reduced. Expressions of oncogenic proteins phospho-Akt and SIRT1 were decreased. 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation.IMPACT STATEMENTWhat is already known on this subject? NNMT is a cytosolic enzyme involved in tumour progression, metastasis and treatment resistance. It was overexpressed in many human malignancies. 5-amino-1-methylquinolinium (5MQ) is a novel small molecule inhibitor of NNMT that has shown promising results in the treatment of obesity and in senescent muscle regeneration. 5MQ has not been tested on the HeLa cervical cancer cell line, previously. What do the results of this study add? In this study, 5MQ was tested on the HeLa cervical cancer cell line for the first time and the molecular changes associated with 5MQ treatment were analysed. 5MQ demonstrated significant anti-proliferative activity on HeLa cells, which displayed morphological signs of apoptosis. Treatment of HeLa cells with 5MQ led to an increase in ZEB1, SIRT1 mRNA while TWIST mRNA was decreased. Phospho-Akt and Sirtuin1 protein expressions were decreased. What are the implications of these findings for clinical practice and/or further research? 5MQ can effectively inhibit HeLa cell proliferation without apparently affecting HEK-293 cell proliferation. 5MQ treatment was associated with a decrease in the expression of phospho-Akt and Sirtuin1 proteins, both of which have been reported to maintain tumour progression. 5MQ can further be investigated and modified for anti-cancer therapy.