Several studies have investigated temporal trends in semen quality in Northern Europe, but none has examined this question in Southern Europe. A prior study conducted in Almeria Province (Southern Spain) reported higher sperm count and concentration among Spanish young men recruited from 2001 to 2002 compared with young men from Northern Europe. The aim of this new study was to examine whether semen quality has changed among Spanish young men in the last decade. In this cross-sectional study, questionnaires and semen samples were collected from 215 healthy young university students from Murcia Region between 2010 and 2011. The 273 men from the Almeria study previously studied were included in a trend analysis of the two populations from Southern Spain. Descriptive statistics were calculated for the Murcia study population and these and semen variables for the Murcia and Almeria study populations were compared. Study methods and population characteristics were similar across the two studies. Therefore, we used multiple linear regression analyses on the combined population (controlling for study centre, age, ejaculation abstinence time, season, smoking, medication during the last 3 months, Body mass index (BMI), presence of varicocoele and prenatal exposure to tobacco) to look for a birth-cohort effect over the combined study period (2001–2011). Sperm concentration and total sperm count declined significantly with year of birth in the pooled analysis (β = −0.04 and β = −0.06, respectively, both p < 0.01). Sperm counts were significantly lower in Murcia study subjects than in the Almeria participants; sperm concentration median (5th–95th) = 44.0 (8.9–129) million/mL vs. 51.0 (5.0–206) million/mL; p < 0.01 and total sperm count = 121 (17.8–400) million vs. 149 (8.0–599) million; p < 0.01. Other semen variables did not differ significantly between the two studies. Our study suggests that total sperm count and sperm concentration may have declined in young Spanish men over the last decade.