Cyclophosphamide used in chemotherapy of cancerous cells was reported to threaten DNA integrity of normal cells like spermatozoa via its ability to generate free radicals. Pre-treatment with antioxidants like N-acetylcysteine was therefore found useful as an adjunct drug against cyclophosphamide-induced cardiac failure and sperm toxicity without interfering with its chemotherapeutic efficacy. In an earlier report from our laboratory, N-acetylcysteine enhanced sperm Histone-Protamine replacement and protected sperm DNA integrity in cyclophosphamide treated male Wistar rats. This suggests a possible interaction between sperm DNA and N-acetylcysteine, however, the nature of the interaction is yet to be elucidated. This study was therefore designed to investigate the molecular interaction between N-acetylcysteine and DNA following cyclophosphamide exposure in-vitro. Herring sperm DNA (1.38×10−3 mol/L), cyclophosphamide (0.6×10−3 mol/L) and N-acetylcysteine (3.58×10−3 mol/L) were prepared freshly and their absorbances were recorded as ADNA, ACYCLO, and ANAC respectively at 260nm. The DNA was then incubated with either cyclophosphamide or N-acetylcysteine and the absorbances were recorded as ADCYCLO or ADNAC respectively. Chromicity change estimated by comparing the sum of ADNA and ACYCLO or ANAC with ADCYCLO or ADNAC respectively were used as indices of molecular interaction. ADNA+CYCLO OR NAC greater than or less than ADNA+A CYCLO OR NAC were considered hyperchromic due to DNA stabilization or hypochromic effects due to DNA fragmentation respectively while unchanged chromicity was considered no molecular interaction. DCYCLO and DNAC were further incubated with NAC and CYCLO respectively and recorded as ADCYCLO+NAC and ADNAC+CYCLO. The sum of ADCYCLO + ANAC or ADNAC + ACYCLO was compared with ADCYCLO+NAC or ADNAC+CYCLO respectively to assess competitive interaction between cyclophosphamide and N-acetylcysteine on DNA. All procedures were repeated 7 times. Data were presented as mean ± SEM and compared by ANOVA at p≤0.05. Hypochromic effect was observed when cyclophosphamide was incubated with DNA alone (ACYCLO=32.44×10-3 ± 0.09×10-3; ADNA= 374.03×10-3 ± 3.10×10-3; ADCYCLO = 384.07×10-3 ± 19.02×10-3) but there was no effect when DNA was pre-incubated with N-acetylcysteine (ADNAC+CYCLO = 412.37×10-3 ± 18.07×10-3). On the other hand, there was hyperchromic effect when N-acetylcysteine was incubated with DNA alone and when the DNA was pre-incubated with cyclophosphamide (ANAC = -117.14×10-3; ADNA= 374.03×10-3 ± 3.10×10-3; ADNAC = 457.93×10-3 ± 19.65×10-3 and ADCYCLO+NAC = 457.77×10-3 ± 19.02×10-3). It was therefore concluded that N-acetylcysteine may proffer a DNA structural stability before cyclophosphamide administration while it could ameliorate DNA damage after cyclophosphamide administration.