Hepatitis E virus (HEV) infection is endemic in Cambodia. However, little relevant data were available and there is no clue if HEV is an emerging or decreasing pathogen in that setting. The aim of our study was to describe temporal trends of anti-HEV IgG and IgM prevalences during the last two decades (1996–2017) in the context of population growth and urbanisation in Cambodia. A total of 2004 human plasma samples collected between 1996 and 2017 were tested for anti-HEV IgG and IgM using the commercial Wantai anti-HEV assays. Overall, the prevalences of anti-HEV IgG and IgM were 41.1% and 2.7%, respectively. Analysis by calendar period showed a decreasing trend of anti-HEV IgG prevalence over the last 21 years. After age- and gender-standardisation, the anti-HEV IgG prevalence rates decreased from 61.3% during the 1996–2000 period to 32.3% during the 2016–2017 period, but no trends were observed for anti-HEV IgM rates, which fluctuated around the overall one. In conclusion, our results suggest that HEV is not an emerging pathogen, but rather seems to circulate less in Cambodia, in particular, in Phnom Penh, since the prevalence of anti-HEV IgG has been significantly decreased during the past two decades.